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Please be advised that this information was generated on 2017-12-05 and may be subject to change. Cerebral visual impairment visual Cerebral from clinic to genetics to clinic from

Cerebral

visual from clinic to genetics Daniëlle G.M. Bosch G.M. Daniëlle impairment

Daniëlle G.M. Bosch

Cerebral visual impairment: from clinic to genetics

Daniëlle G.M. Bosch The thesis can also be downloaded from: http: http://www.e-pubs.nl?epub=d.bosch login: d.bosch password: pyzwyk

The studies presented in this thesis were performed at Bartiméus, Institute for the Visually Impaired, Zeist, The Netherlands, and at the Department of Human Genetics, Radboud university medical center, Nijmegen, The Netherlands.

The research of D.G.M. Bosch was financially supported by Stichting ODAS, Vereniging Barti- méus-Sonneheerdt (5781251), and, contributed through UitZicht; Oogfonds and Landelijke Stichting voor Blinden en Slechtzienden (2014-9).

ISBN: 978-94-6169-776-9

Layout and printing: Optima Grafische Communicatie, Rotterdam, The Netherlands. Cover design: Optima Grafische Communicatie, Rotterdam, The Netherlands

Copyright © D.G.M. Bosch, 2015. All rights reserved. No parts of this thesis may be reproduced, stored in a retrieval system of any nature, or transmitted in any form or by any means, without prior written permission of the holder of the copyright. Cerebral visual impairment: from clinic to genetics

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 3 februari 2016 om 10.30 uur precies

door

Daniëlle Gerda Maria Bosch

geboren op 10 juni 1983 te Tegelen Promotor: Prof. dr. F.P.M. Cremers

Copromotoren: Dr. F.N. Boonstra (Bartiméus, Zeist) Dr. L.B.A. de Vries

Manuscriptcommissie: Prof. dr. B.G.M. van Engelen (voorzitter) Prof. dr. C.C.W. Klaver (Erasmus MC) Prof. dr. ir. S.M. van der Maarel (LUMC) Table of contents

Chapter 1: Introduction 7

1.1 Anatomy of the visual system 9 1.2 Visual development 12 1.3 Cerebral visual impairment 15 1.4 Introduction into genetics 24 1.5 Scope, methods and outline of the thesis 28

Chapter 2: CVI: acquired and genetic causes 33

2.1 Low vision due to cerebral visual impairment: differentiating between acquired and 35 genetic causes 2.2 Chromosomal aberrations in cerebral visual impairment 93

Chapter 3: Identifying genes for CVI 129

3.1 Novel genetic causes for cerebral visual impairment 131 3.2 Cerebral visual impairment and intellectual disability caused by PGAP1 variants 173 3.3 NR2F1 mutations cause optic atrophy with intellectual disability 189

Chapter 4: Overall results of the CVI study 205

4.1 Characteristics and results of the cohort 207 4.2 Conclusions and implications 211

Chapter 5: General discussion and future perspective 213

5.1 Challenges in variant interpretation 215 5.2 CVI: a separate entity? 218 5.3 An outlook 219 5.4 Final remark 221

Appendices 223

Reference list 225 Summary 246 Samenvatting 249 List of authors and affiliations 252 Dankwoord/Acknowledgements 256 Curriculum Vitae 258 List of publications 259 List of abbreviations 262

Chapter 1

Introduction

1.1 anaToMy of The VIsual sysTeM

When light enters the eye, the photoreceptors in the retina transform the light into an electric signal. There are two types of photoreceptors: cones and rods. The three diff erent types of Chapter 1 cones give us the ability to see colours, and they are mainly localized in the macula (Figure 1). The central part of the macula, the fovea, consists of the highest concentration of cones and is responsible for sharp vision. In contrast, the rods dominate the (mid-)periphery of the retina and are used in peripheral vision.1 They are the primary source of visual information in the dark. The optic signal which the photoreceptors produce is transported via the bipolar cells to the retinal ganglion cells. There are three main types of retinal ganglions cells; the midget ganglion cells (project to parvocellular neurons, P-system, which is the largest system), the parasol cells (project to magnocellular neurons, M-system) and the bistratifi ed cells (project to koniocellular neurons, K-system) (Figure 2).2,3 The axons of the retinal ganglion cells are the fi bres of the optic nerve, and mainly terminate at the lateral geniculate nucleus (LGN) of the thalamus (retino-occipital visual pathway). The LGN is highly organised: layer 1, 4 and 6 receive axons from the contralateral nasal retina, whereas 2, 3, 5 receive axons from the ipsilateral temporal retina.3 The magnocellular neurons are present in layer 1 and 2, the parvocellular neurons in layer 3-6 and the koniocellular neurons are present in between.3 The M-system is characterized by low spatial resolution with rapid transmission of nerve impulses, and is therefore important for movement processing.3 In contrast, the P-system is characterized by high spatial resolution but slow transmission of nerve impulses, and is mainly involved in object processing.3 The function of the K system is less clear, but might be involved in colour processing.2,3 In addition to the retino-occipital pathway, there are several other essential but less studied visual pathways including the retino-collicular pathway (important for refl exive eye move-

Light Light

Cornea

Lens Optic nerve Macula Ganglion cell Retina Bipolar cell Photoreceptor (cone) Photoreceptor (rod) figure 1: a cross section of the human eye with a schematic enlargement of the retina Figure derived and adapted from http://webvision.med.utah.edu/book/part-i-foundations/simple-anatomy-of-the-retina/.

9 Chapter 1

LGN Koniocellular Retina Bistrati ed Parvocellular

Midget Magnocellular

V1 Parasol

2/3 Koniocellular 4A 4B 4Cα Magnocellular 4Cβ Parvocellular 5

Figure 2: An overview of the P-, M- and K-system in the retino-occipital visual pathway Midget, parasol and bistratified ganglion cells are well characterized and have been linked to parallel systems that remain anatomically separate through the lateral geniculate nucleus neurons (LGN) and into the primary visual cortex (V1). Midget ganglion cells project to parvocellular layers of the LGN (P-system), and parasol ganglion cells project to magnocellular layers (M-system). Subsequently, both project to layer 4 of the primary visual cortex. Bistratified ganglion cells project to koniocellular layers of the LGN and on to layer 2/3 (K-system). Although these ganglion cell types are numerically dominant in the retina, many more types are known to exist and are likely to subserve important parallel systems that are yet to be identified. (Adapted and printed by permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience,3 copyright 2009.) ments, motion discrimination and non-conscious perception of emotional stimuli, mainly M-system), the retino-pretectal pathway (serve the pupillary reflex), the retino-hypothalamic visual pathway (circadian rhythms), and the accessory optic system (head and gaze orienta- tion and slow eye movements) (Figure 3).4-7 Following the retino-occipital visual pathway, the LGN project via the optic radiations mainly towards layer IV of the primary visual cortex in the occipital lobe of the brain.8 The primary cortex is organised into ocular dominance columns (ODC), meaning that bands of cortical tissues alternately receive input from the left or the right eye (Figure 4).9 Because of the partial crossing of the optic nerve at the optic chiasm, the primary cortex receives visual information from both eyes of the same side, thus the right visual field is processed by the left hemisphere and vice versa.1

10 Introduction

Optic Optic tract nerve Chapter 1

Optic Hypothalamus chiasm

Lateral geniculate Pretectum nucleus

Optic radiation Superior colliculus

Primary visual cortex figure 3: an overview of the visual pathways, from the eye to the primary visual cortex Figure derived and adapted from https://www.studyblue.com.

Temporal Primary visual cortex layer 4 retina Ipsilateral

Nasal Contralateral retina

Nasal Ipsilateral retina LGN

Contralateral Temporal retina figure 4: segregation of eye-specifi c information at the early stages of visual processing In mammals with binocular vision, the nasal portion of one retina encodes the same part of the visual world as the temporal portion of the other retina. The axons of retinal ganglion cells from the nasal portion of each retina cross the optic chiasm and project to the same LGN as the axons from the temporal portion of the other eye. These projections form discrete, eye-specifi c LGN layers. The projection from the LGN to layer 4 of the primary visual cortex maintains this eye-specifi c segregation by terminating in eye-specifi c patches that are the anatomical basis for ocular dominance columns. Ocular dominance columns can therefore be considered to correspond to an eye (left or right) or a retinal location (nasal or temporal). (Adapted and printed by permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience,9 copyright 2002.)

11 Chapter 1

Within the primary cortex there are connections between the diff erent neurons and layers, suggesting that the input from the diff erent systems (P-, M- and K-system) is mixed, before the visual information is projected to other brain areas.3 About 50 years ago the concept of two main visual streams from the primary cortex was proposed.10-12 The dorsal (‘where/how’) pathway, from the primary visual area to the parietal lobe, is mainly responsible for processing spatial information necessary for planning and programming of motor actions. The ventral (‘what’) pathway, from the primary visual area to the temporal lobe, is mainly responsible for conscious visual perception, visual recognition and visual memory.13,14 Although this concept is widely used, it is probably an oversimplifi cation, because there are many cortico-cortical connections between the diff erent brain areas processing visual information.12. This complex combination of the input of the diff erent visual pathways and systems, together with the cortico-cortical connections leads to the perception and interpretation of the visual input.

1.2 VIsual DeVeloPMenT

Prenatal development of the visual system The visual system develops from the forebrain, which is already recognizable in the third week after fertilization.15-17 The eye development starts with the formation of the optic vesicle at day

A B C D Surface ectoderm

Mesenchyme Lens Neural Retinal pigmented Neuroepithelium vesicle Lens retina epithelia

Optic vesicle

Optic stalk

Lens Bilayered Photoreceptor cells placode optic cup Interneurons Ganglion cells figure 5: Development of the human eye A and B) When the optic vesicle reaches the surface ectoderm, it invaginates and the adjacent surface ectoderm starts to develop into the lens. C and D) The inner and outer wall of the optic cup give rise to the neural retina and the retinal pigmented epithelium. The neural retina develops into an outer neuroblastic layer, from which the photoreceptors derive, and the inner neuroblastic layer, from which the bipolar and retinal ganglion cells arise. The axons of the retinal ganglions cells grow into the optic stalk. (Adapted and printed by permission from Macmillan Publishers Ltd: Nature,18 copyright 2011.)

12 Introduction

A Optic nerve & tract B G

C Chapter 1 Optic radiation D E Cortex F

0 4 8 12 16 20 24 28 32 36 2 4 6 8 Weeks after fertilization Birth Postnatal months figure 6: Main periods in the development of the visual system A) Between 6th – 14th weeks of development the axons of the retinal ganglion cells grow into the optic stalk towards the thalamus and synaps with the LGN in the thalamus.15,16 B) Around 16 weeks about 3,7 million axons are present, but about two thirds will degenerate and 1,1 million have an established connection at birth.19 C) From the 15th week onward the thalamocortical axons grow into the subplate and synapse with the subplate neurons.21,22 D) Between the 24th until the 32nd week the subplate connection disappears and the thalamocortical axons grow into layer IV of the cortex to establish synapses with the layer IV neurons.21 E) Subsequently, the maturation and refi nement of the thalamo- and cortico-cortical connections will start.21 F) From the 30th week onwards the cortex starts to myelinate.24 G) Myelination of the optic nerve takes largely places between the 32nd week and 7 months postnatal.25

22.15 When the optic vesicles make contact with the surface ectoderm, they invaginate to form the optic cups. The stem of the optic vesicle narrows to form the optic stalks (Figure 5).15,16 Simultaneously, the adjacent surface ectoderm starts to develop into the lens.15 The inner and outer wall of the optic cup give rise to the neural retina and the retinal pigmented epithelium.15 The neural retina further develops into an outer neuroblastic layer, from which photoreceptor cells derive, and the inner neuroblastic layer, from which the bipolar and retinal ganglion cells arise.15,16 From the sixth week of development the axons of the retinal ganglions cells grow into the optic stalks, which will transform into the optic nerves (Figure 6).15,16 The axons from both eyes join and partly cross at the optic chiasm and subsequently synaps with the LGN in the thalamus.15 Of the 3.7 million axons that are initially present (16th week), only 1.1 million will achieve an established connection at birth; the other axons will degenerate.19 In the fi rst weeks after fertilization also the cortex starts to develop. From the ventricular zone, adjacent to the ventricles, neurons proliferate and migrate to the correct layer (sixth week). They migrate initially to the most inner layer and fi nally to the most outer layer (layer I).8,20 One transient layer during development is the subplate zone. The thalamocortical axons fi rst synaps with the subplate neurons (15th -24th week), which at their turn connect with the neurons of layer IV.21,22 After this ‘waiting period’ in the subplate the thalamocortical axons grow into layer IV themselves and the subplate neuron connection disappears.20,23

13 Chapter 1

Postnatal development of the visual system Although at birth the main structures are present, the visual system is still immature. The major part of the postnatal development takes place in the first period after birth, however the visual system does not reach full maturity before adulthood. For example: the fovea reaches maturity at 4 years, whereas myelination has its peak in the first year after birth, but continues into the third decade of life.26-28 For further development visual input at the right moment is essential. This is illustrated most clearly by the disorder amblyopia: an unilateral or, less commonly, bilateral reduction of the best corrected visual acuity (VA) that cannot be attributed directly to the effect of any structural abnormality of the eye or the posterior visual pathways.29,30 When a child is a few months old equal and sufficient visual input of both eyes is necessary to obtain normal VA. This lasts until the age of eight and is called the critical period. When one eye is not or hardly used due to strabismus, unequal refractive errors or cataract, amblyopia occurs.29,31 Amblyopia can be recovered by eliminating the underlying cause and patching the (better seeing) eye. The earlier this intervention starts the better the outcome. However, partly improvement of the VA by using patching can still be achieved in adolescence.31,32 Recently, binocular dichoptic training and brain stimulation was used in amblyopic adults with promising results.33-36 As a result of the immature system, a newborn has limited vision. This improves rapidly; at birth VA is between 20/1200-20/400 (0.015-0.05) and fixation is intermittent present.37 From

32.00 20/19

16.00 20/38

8.00 20/75

4.00 20/150

2.00 20/300

1.00 20/600 Acuity (cycles/degree) Acuity ‘equivalent’) (Snellen Acuity 0.50 20/1200

0.25 20/2400 0 6 12 18 24 30 36 42 48 Age (months)

Salomao & Ventura - 90% tolerance limit with 95% con dence, Invest Ophthalmol Vis Sci, 1995 Courage - 99% prediction limit, Optom Vis Sci, 1990 Courage - 95% prediction limit, Optom Vis Sci, 1990

Figure 7: Development of visual acuity Development of VA measured binocular by Teller acuity cards (grating acuity at 55 cm), and Snellen ‘equivalent’ (optotype acuity at 6 meters). (Figure derived and adapted from Teller acuity cards™ II handbook, 2005)

14 Introduction two to three months onwards a child fixates well on human faces or nearby toys, and can follow a moving object smoothly.37,38 When the brain and the fovea mature the VA improves. Because different tests assess different visual aspects, such as forced preferential looking and 30,32,39,40 measurement of VA with optotypes, the VA depends on the test used. For the different Chapter 1 methods for VA measurement age norms have been defined (Figure 7).30,39,40 The difference between single VA with optotypes and line VA is called crowding, defined as the impairment of the ability to recognize objects in clutter, and can persist until adolescence.41,42 Nevertheless, a near crowding ratio >2 (single optotype acuity divided by linear acuity) at all ages or a distance crowding ratio >2 after the age of 6 years is a reason for further investigation.43 The visual fields expand rapidly after birth, starting from approximately 30% of adult values at 2 months, to 75%-80% at 8 months and 100% at 2 years.32,44

1.3 Cerebral visual impairment

The disorder cerebral visual impairment Vision loss due to pathology of the brain was first recognized in adults and was called cortical blindness, for reviews see Hoyt and Good et al.45,46 However, in children with brain pathology it was noticed that the majority was not completely blind, so the term cortical visual impairment was introduced.45 This name, though, is still misleading, as in many children not only the cortex is involved. Therefore, the preferred name is cerebral visual impairment, but cortical visual impairment is still commonly used.45,47 For this thesis the term cerebral visual impairment abbreviated as CVI will be used. CVI was first defined as “a neurological disorder resulting in bilateral impairment of VA by damage to the central nervous system, meaning VA is reduced as a result of a non-ocular disease”.46,48 During the beginning of the 21st centuries it was noticed that CVI could also lead to other visual disorders without VA loss and the definition was modified: “CVI includes all visual dysfunctions caused by damage to, or malfunctioning of, the retrochiasmatic visual pathways in the absence of damage to the anterior visual pathways or any major ocular disease”.49,50 Problems with processing and integrating of visual information are also known as higher functioning CVI, visuoperceptual disorders or cognitive visual dysfunction.48,51,52 Although amblyopia is also a kind of CVI, in general amblyopia is not meant when speaking of CVI.29 CVI is a major and increasing cause of visual impairment in children in Western countries. This is probably the result of better care for curable eye disorders (e.g. cataract), more awareness of CVI, and a higher survival rate for preterm born children and children with multiple congenital abnormalities and/or severe neurological disorders.53 CVI is responsible for almost one third of low vision in children, a VA of ≤0.3 and/or visual field <30.53,54. In a group of children with a VA ≤0.1 and/or visual field <20°, CVI accounted for 18%-48% of the impair-

15 Chapter 1 ment.55-57. The number of children with CVI without low vision is currently unknown. In this thesis, the focus will be on CVI with low vision (VA ≤0.3 and/or visual field <30°).

Ophthalmological investigation and findings The most important ophthalmological examinations and the possible results for patients with CVI will be discussed. The majority of the studies of CVI are performed in individuals with perinatal damage. Whether the results can be extrapolated to CVI due to other causes is unknown.

Fixation, eye movements and eye alignment An important aspect of the ophthalmological examination is observation: does the patient fixate at and follow the examiner and interesting objects. Fixation can be unstable in patients with CVI, such as frequent loss of fixation, the inability to sustain fixation or no fixation at all.58,59 The fast eye movements towards a stimulus, reflexive saccadic eye movements, can be impaired.59 In addition, the reaction time to fixation can be delayed.60 In CVI patients devia- tions of conjugated gaze can be observed. These are moments of uncontrolled wandering eye movements mainly made upward or sideward.59,61 Furthermore, smooth pursuit can be saccadic or completely absent in CVI.52,59 Although CVI was previously only diagnosed in the absence of nystagmus, it is now commonly accepted that nystagmus can be one of the ocular features.47,59,61,62 Strabismus can be present in patients with CVI. In patients with cortical damage exotropia is more common, whereas in patients with subcortical damage esotropia is more common.61 Fluctuating eso-, ortho-, and exotropia (dyskinetic strabismus) can also be observed in patients with CVI.61 During observation abnormal visual behaviour can be seen, including looking away from the target while reaching to it, looking next to the target, not watching when listening, and staring into lights.46,58

Visual acuity VA measurement in children under the age of 2 years is often possible by using forced preferential looking or visual evoked potentials (VEP).30,37,63 In VEP measurement the electric potential in the visual cortex initiated by a brief visual stimulus is recorded. In CVI the VEP vernier acuity measurement correlates best with forced preferential looking at Teller acuity cards (TAC) based on grating acuity.64 However, the sweep VEP might be predictive for the future grating or recognition acuity.65 Grating acuity cards (e.g. TAC) are based on the reflex of people to look at contrasting patterns instead of a homogeneous target (Figure 8).38 Tests based on object recognition, such as the LH test or Landolt C test can be used in older children.66 From 6 years onwards acuity can often be measured by Snellen acuity charts. For the LH, Landolt C and Snellen test it is possible to measure single VA or line VA (in a row), which

16 Introduction

A B Chapter 1

figure 8: Diff erent charts to measure the visual acuity A) Teller acuity cards. B) LH test uncrowded. C) LH test crowded. is useful to detect crowding. In crowding measurement there is an infl uence of test design: a chart with fi xed intersymbol spacing should be used, since charts with proportional intersymbol spacing do not reveal diff erences between normally sighted and visually impaired children.67 The acuity defi ned by VEP or TAC is a grating acuity and cannot be compared to a recognition acuity, as the tests are based on diff erent aspects of vision. For the purpose of communication often a Snellen ‘equivalent’ is mentioned. In all children and in persons with intellectual disability (ID) the VA test should be related to the developmental level of the person. For example in an adult with CVI and a severe intellectual disability the TAC can be the only method to assess the VA. However, in patients with impaired refl exive saccadic eye movements or vertical visual fi eld defects the TAC cannot always be used. In patients with CVI the VA can range from blindness to normal, and can improve over time.50,68,69 There is a inverse correlation between the severity of perinatal damage and the degree of acuity impairment.70,71 Crowding is a feature of the immature visual system, low visual acuity and CVI.67 It is present in about half of the CVI patients.72-74 One other characteristic of CVI can be fl uctuating visual performances and attention, which could hamper the measurement of VA.46,58

Visual fi elds Visual fi eld measurement by perimetry such as the Goldmann is often not possible under the age of 7 years.37 Nevertheless, it is possible to get information about the visual fi elds in younger persons by using a confrontational method with white Stycar balls on a stick (Figure 9).75 A slightly modifi ed version of this method, the BEFIE test, was shown to give reliable results.76

17 Chapter 1

figure 9: Two stycar balls on a stick for examining the visual fi eld The patient is asked to fi xate at one stycar ball, that is held in front of the patient. Another person stands behind the patient and moves the second Stycar ball with a wide circle into the visual fi eld of the patient. A refl exive saccadic eye movement can be observed when the second Stycar ball is noticed.

In patients with CVI the visual fi elds are often impaired, which can consist of homonymous hemianopia, upper and lower visual fi eld, or constriction of the visual fi eld. Visual fi eld defects are more severe when the abnormalities in the posterior visual system are more extensive. Sometimes a clear correlation of the visual fi eld with the brain damage can be seen, for example an occlusion in the middle cerebral artery or anterior cerebral artery may lead to hemianopia.77,78

Slit lamp examination, funduscopy and refraction Slit lamp examination sometimes reveals minimal nystagmoid eye movements. It is also important to investigate the anterior segment (e.g. to detect corneal damage or cataract). With funduscopy, after pupil dilation, it is possible to have a look at the retina and evaluate the fovea and the optic nerve. Young children can be diffi cult to examine, as they only want to fi xate on the light or don’t want to look at the light at all. The latter can be caused by photophobia, which can be present in CVI patients.79 In patients with CVI a pale optic disc or a pale segment of the optic disc can be seen. A small optic disc or a more pronounced excavation is reported in patients with CVI due to perinatal problems.80,81 The presence of refraction errors needs to be evaluated. When the pupils are dilated refraction can be examined objectively with a hand-held autorefractor or by retinoscopy.

18 Introduction

For this measurement steady fixation is necessary, which sometimes lacks in patients with CVI. Hypermetropia is a normal phenomenon during childhood, and can be corrected by accommodation. In CVI patients incomplete accommodation with persistent hypermetropia 82 51,79 occurs. However, myopia and astigmatism can also be present in CVI patients. Just like Chapter 1 normally sighted person, refraction errors are common and important to correct in CVI patients. However, it may be more difficult to get the child accustomed to glasses.52

Electroretinography and optical coherence tomography Electroretinography (ERG) is used to investigate the function of the cells in the retina, among others the photoreceptors. An electrode is put on the forehead and wire or lens electrodes are placed on the anesthetized eye to measure the flash induced responses. An ERG is a demanding examination and can only be performed in cooperative older children or under anesthetics.30 ERG abnormalities can be found in retinopathies. In individuals with CVI without other ocular deficits the ERG can be normal. However, in optic atrophy, which can be seen in CVI as a result of retrograde degeneration of optic nerve fibres, the ERG may be abnormal. Optical coherence tomography (OCT) produces a cross sectional image of the retina, macula and optic disc by using infra-red light. Patients should be able to fixate and willing to put their head in the standard. Aberrant OCT images may be present in CVI, because the optic disc can be abnormal. However, to our knowledge, no OCT studies have been performed in a CVI cohort.

Higher visual functioning Disorders in higher visual functioning are sometimes the only features of CVI.50 For example, a patient can have problems with the detection of movements, difficulties in recognition of faces, expressions or objects or inability to differentiate a floor boundary. Inability to select relevant visual information in a busy scene, crowding, can also be a deficit of higher visual functioning. For a first screening of higher perceptual deficits several questionnaires have been developed that can be filled in by parents or caretakers.72,83,84 More detailed neuropsychological tests, such as the L94 visual perceptual battery, can be helpful to gain more insight in specific higher perceptual deficits in the individual.85,86 However, not all questionnaires and batteries are validated in toddlers, persons with intellectually disability and/or in patients with a VA ≤0.3, so these questionnaires may not be suitable for these persons.87

Comorbidities of CVI The malfunctioning or damage of the brain is mostly not limited to the visual system. There- fore, additional features like epilepsy, delayed development and intellectual disability can be present.53 Sometimes CVI can also be accompanied by abnormalities of other organ systems. Thus CVI can occur in isolation or be part of a syndrome.

19 Chapter 1

Diagnosing CVI CVI is an umbrella diagnosis of any kind of malfunctioning of (parts of) the visual system behind the eye, leading to many different presentations.88 This makes it difficult to formulate clear criteria for the diagnosis. CVI is often diagnosed when no ocular disorder can explain the visual impairment, so called diagnosis per exclusionem. The diagnosis evolved over time, and additional conditions were proposed to be required, such as recognizable brain damage on imaging, low VA, absence of nystagmus, or abnormalities at detailed neuropsychological investigations (personal experience).46,62,89 Probably the most comprehensive criteria nowadays are published by C. Roman and others: “(1) an eye examination that cannot fully explain the child’s use of vision; (2) a history or presence of neurological problems, even when the child’s brain-imaging studies may appear normal; and (3) the presence of the behavioural or visual responses that are collectively associated with CVI.”90

Differential diagnosis If an infant or young child presents with a bilaterally impaired visual acuity, the most important diagnoses to exclude are cataract, uncorrected refraction errors, retinopathies, albinism, and delayed visual maturation.39,53 Most of them can be excluded by thorough ophthalmological examination. The most important and more challenging differential diagnoses are discussed below.

Leber congenital amaurosis Children with Leber congenital amaurosis (LCA) often push or rub their eyes with a finger or fist, the oculodigital sign. The VA range from 0.1 (rare) to no light perception, and the eye movements can be wandering. In addition, patients can have nystagmus, high hypermetropia, photophobia and poor or absent pupil responses. The fundus can appear normal in the first year after birth, but later pigmentary changes, vascular narrowing and/or optic disc pallor can be observed. Pattern and flash VEP are mostly absent, but diagnostic is the ERG, which shows extremely attenuated or absent responses.39,91.

Delayed visual maturation Delayed visual maturation (DVM) is a disorder in which the infant shows poor visual functioning for his/her age, without any ocular disorder. It shows strong resemblances with CVI, however in DVM the vision improves to normal, generally within 6-12 months, but longer periods have been reported.39,92,93 Although the vision fully recovers, these children have a higher risk on developmental disorders, and vice versa DVM can occur in children with developmental delay.89,91,94,95 Some authors see DVM as a mild form of CVI.91 Nevertheless, the boundary between CVI and DVM still needs to be determined. Some suggested criteria for DVM are a normal brain MRI, normal flash VEP for age and full recovery of vision.91,93,94

20 Introduction

However, this is challenged by reports in which children with DVM have persistent deficits or VEP abnormalities.94,95

Nystagmus and other motility disorders of the eye Chapter 1 Infantile nystagmus is often a feature of an underlying disorder, such as albinism or congenital stationary night blindness.91,96 In infantile ‘idiopathic’ nystagmus, thus without an underlying ocular or neurological disorder, the VA can be impaired but is in general above 0.3.96 In addition, there are no co-morbidities and frequently there is a family history of nystagmus. Congenital oculomotor apraxia can be difficult to distinguish from CVI.47 The condition is featured by the inability to fix and follow moving targets, which could give the impression that the child is blind. However, an optokinetic nystagmus, induced by rotation, is intermittent or absent in congenital oculomotor apraxia and older children use head movements and head thrusting to fixate and follow.97

Optic nerve abnormalities The VA in patients with optic nerve abnormalities can vary from normal vision to blindness. Other features like visual field defects, nystagmus or strabismus can be present.92,98 Two optic nerve abnormalities relevant for the differential diagnosis of CVI are optic atrophy and optic hypoplasia.47,99 It can be difficult to define whether it is a developmental disorder of the optic nerve (optic hypoplasia), an endogenous defect resulting in increased apoptosis of optic nerve fibres (primary optic atrophy), or transsynaptic retrograde degeneration of the optic nerve fibres (secondary optic atrophy, which can be present in CVI). First of all there is a discrepancy between the ophthalmological and pathological definition of hypoplasia and atrophy. Optic hypoplasia is defined by pathologists as an underdevelopment, which means less fibres in the optic nerve.100 Optic atrophy is defined as degeneration, which means loss of fibres.101 Ophthalmologist base the terminology on the characteristics of the optic disc, a small optic disc is called hypoplasia and a pale optic disc is called is atrophy.39 In patients with early gestational injury optic hypoplasia has been reported by ophthalmologists.61,102 However, in these patients the smaller optic nerves are probably due to an increase of the normal apoptosis of axons in an early stage of development, thus atrophy might be a more appropriate term.99,103 Optic nerve hypoplasia is also seen in combination with other abnormalities such as anterior eye defects and midline defects, including corpus callosum agenesis, hypothalamic or pituitary dysfunction.98 The pathophysiology underlying the small optic discs in these disorders is largely unknown. It may be a primary failure of the retinal ganglion cell to develop or, as now more commonly believed, due to a secondary apoptosis of the axons as a result of interference of normal axonal migration within the visual pathways.92 Secondly, it can be difficult to define whether the optic atrophy is primary or secondary. Secondary optic atrophy develops due to transsynaptic retrograde degeneration, which can occur when the retrochiasmal parts of the visual system are malfunctioning, for ex-

21 Chapter 1 ample in gestational injury or other brain abnormalities.92,104,105 The aspect of the optic disc in transsynaptic retrograde degeneration is indistinguishable from primary optic atrophy. Therefore, discriminating between (pre-) chiasmal (primary optic atrophy) and retrochiasmal disorders (secondary optic atrophy, which can be present in CVI) can be challenging and this implicates that a commonly used definition for CVI is not always applicable in clinical practice: ‘CVI includes all visual dysfunctions caused by damage to, or malfunctioning of, the retrochiasmatic visual pathways in the absence of damage to the anterior visual pathways or any major ocular disease’.49 Nevertheless, the absence or presence of other ophthalmological features (e.g. normal eye movements) and the history (e.g. late onset with detoriation) can often discriminate between CVI and other forms of optic nerve abnormalities.99

Therapy in CVI It is important to make the patient, his family, and caretakers aware of CVI, in order to make them understand the sometimes strange visual behaviour, e.g. not noticing objects in a busy environment. Depending on the level of impairment, this can require specific adjustments as pointed out in Table 1. For instance, it can be better to use pictograms instead of photographs as communication aid. In general, it is important to structure and simplify the visual environment. To train the patient, the situation can slowly become more complex, guided on the level of the patient. Furthermore, it can be necessary to teach the patient about the meaning of visual information. For instance, let the patient feel with hands and feet what the differences in height are between the carpet and parquet. Furthermore, vision strategies can be used. Persons with lower visual field defects for example, can be learned to scan the whole ground by head and eye movements. In addition, trainings have been developed to stimulate

Table 1: Examples of impairments, strategies and trainings for CVI Impairment Examples of strategies Examples of training Auditory stimuli predominate Structured acoustic circumstances (e.g. put of the radio) Motility disorder of the eyes Structured visual input Guided search training Visual impairment in general Optimize lightning circumstances Low vision aids Crowding Structured visual input (e.g. plain place Gradually make the situation more mats or agree upon a specific place on the complex school yard) Visual field defects Present objects in the remaining visual field Teach to use head movements Difficulties with abstract figures Use photographs Training in which line drawings become a real object Difficulties with estimating height on Remove colour differences on a flat ground Experience and gradually make the different coloured grounds and highlight stairs with coloured rims situation more complex Facial expressions Support the expression with more emotion From exaggerated expressions in the voice. or emoticons to more subtle expressions

22 Introduction fixation, following, and visual recognition, and to reduce crowding.42,106,107 Although, it is plausible that these trainings will improve vision, just like physiotherapy improves motor development, it is hardly been formally investigated in CVI.42 Chapter 1 Aetiology of CVI Acquired causes In brief, all events that cause brain injury prenatal, perinatal or postnatal can lead to CVI. The disorders with a high risk on CVI and several illustrative examples will be discussed below from prenatal to postnatal events. Prenatal infections, such as congenital CMV infections, could lead to CVI. About 0.6% of the newborns are infected by CMV, of which 10% is symptomatic at birth.108 Especially these symptomatic newborns are at risk to develop visual impairment due to CVI.109 Other prenatal events can be maternal drug abuse or twin-twin transfusion syndrome.52,110 A well recognized cause of CVI are perinatal problems. The risk on perinatal problems increases with a decrease of the gestational age at birth and a lower birth weight.111,112 In two studies the risk on CVI with low vision was 1-2% in premature born children (birth weight <1500g or gestational age <27 weeks).113,114 The perinatal problems underlying CVI may consist of periventricular leukomalacia, brain haemorrhages, sepsis, and neonatal hypoglycaemia. Postnatal risk factors include severe head trauma, infections of the brain, severe uncontrolled epilepsy or hydrocephalus.52,115-118

Genetic causes Till recently, in the larger CVI aetiology studies, varying from 100 to 170 persons with CVI, minimal attention is paid to the genetic disorders involved.51,79,119 For some genetic syndromes the affected individuals are systematically investigated for deficits in higher visual functioning by neuropsychological test. For example in Noonan syndrome, Williams syndrome, and Prader-Willi syndrome; higher visual disorders are diagnosed, but impairment of VA has not been reported.120-122 However, many reports of genetic syndromes and CVI are case studies in which the ophthalmological examination (especially VA), the perinatal condition, or the molecular diagnosis are not reported in detail. Therefore, it is difficult to come up with a complete and reliable list of genetic disorders in which CVI with low vision is part of the phenotype. Nevertheless, based on the sporadic literature, the genetic causes underlying CVI can be divided into three groups. In the first group CVI is indirectly caused by the genetic defects, such as disorders that increase the risk of stroke or brain tumours; for instance mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), protein C deficiency, tuberous sclerosis.62,123-125 The second group consists of progressive neurodegenerative disorders, such as Leigh syndrome or X-linked adrenoleukodystrophy.62 The third group is

23 Chapter 1 the group of the non-progressive disorders. CVI is for example reported in chromosomal disorders, such as Down syndrome and 1p36 deletions, and monogenetic disorders, such as congenital disorders of glycosylation.126-132

1.4 InTroDuCTIon InTo GeneTICs

Genetics The fi rst large contribution to our understanding of inherited disease nowadays was made by G. Mendel (1822-1884) who reported dominant and recessive inheritance in 1865.133 Forty years later it was proposed that the chromosomes in the nucleus were the bearers of this heredity,134,135 and it was thought that a human being had 48 chromosomes. In 1956 the correct number of 46 was established (Figure 10).136 Only a few years later, in 1959, the fi rst numeric chromosomal disorder was identifi ed, trisomy 21 or Down syndrome,137, and when G-banding karyotyping was introduced the resolution to detect aberrations became 5-10 Mb.138 In the years that followed other techniques to investigate our chromosomes were developed and it was shown that also submicroscopic aberrations (<5 Mb) could lead to neurodevelopmental disorders.138 After targeted techniques, such as fl uorescence in situ hybridization (FISH) or unbiased techniques such as multiplex ligation-dependent probe am- plifi cation (MLPA) telomere screening, the genome-wide microarrays were introduced.139,140 The resolution improved over time up to the detection of aberrations as small as 20 kb cur-

A Chromosome B C

Mother

Father

Cell nucleus Child

D C T G C T A C G C E C

Mother Mother Gene Father Father Gene DNA Child Child figure 10: Diff erent methods to perform genetic investigations A) Schematic presentation of the genetic material. B) Karyogram revealing trisomy 21. C) Microarray led to the identifi cation of a de novo deletion on 4q28.3q31.1 of 2.73 Mb. D) Sanger sequencing reveals a de novo c.755T>C variant in NR2F1. E) Whole exome sequencing identifying a de novo c.3646G>A variant in GABRB2.

24 Introduction rently. Copy number variants (CNVs) are present in healthy and affected persons. On group level, patients with intellectual disability (ID) and autism have more and larger CNVs, which arise more often de novo and are less frequent in the global population compared to healthy 141-143 individuals. However, these statistics are not useful on individual level, because they are Chapter 1 not discriminative. For the interpretation whether a CNV is pathogenic, workflows have been developed.144-146 These workflows include the following aspects: comparing the phenotype of persons with a similar CNV by using control and patient datasets (e.g. Database of genomic variants (DGV), DECIPHER and ECARUCA (https:// http://dgv.tcag.ca/, https://decipher.sanger. ac.uk/ and http://umcecaruca01.extern.umcn.nl:8080/ecaruca/),147-149 establishing the origin of the CNV (de novo or inherited from a healthy or affected parent), and investigating the genes involved. With the use of these microarrays a causal chromosomal aberration could be detected in 10-20% of the patients with developmental delay/ID, autism spectrum disorders, and/or multiple congenital anomalies.150-152 This had led to the identification of many new microdeletion and microduplication syndromes reviewed by Van Bon and Watson et al.153,154 In the fifties not only the exact number of chromosomes was identified, but also the double helix structure of DNA was discovered.155 In the following 10 years the relation between the sequence of the bases (adenine, cytosine, guanine, and thymine) and encoded proteins became clear, which made it possible to predict the effect of a nucleotide variant.156 The first disorder in which a pathogenic amino acid change could be proven was sickle cell anaemia in 1956.157 The next major step was the development of a method to investigate the sequence of nucleotides by F. Sanger in 1977, called Sanger sequencing.158 This initiated a project to sequence the entire genome: the Human Genome Project, which was a collaborative effort of 20 institutes and started in 1990 and was declared finished in 2003.159,160 It turned out that the sequence of DNA between humans is 99% identical,161 implicating that there are millions of variations in the nucleotides. The majority of these variants are situated in the non-coding part of the DNA, but thousands of variants are present in genes. Most variants have no or only a mild effect on the phenotype and contribute to the fact that everybody is a bit different. However, some variants do have severe phenotypic consequences. For example, in ophthalmological disorders, mutations in NDP lead to Norrie disease, characterized by a specific early onset retinopathy, hearing loss, ID, and behavioural abnormalities.162 Based on the phenotype it is now known which gene has to be sequenced, but for other disorders, such as non-syndromic retinitis pigmentosa many genes (>75) can be mutated (https://sph.uth.edu/Retnet/sum-dis.htm accessed on July 2015). To test all genes one by one by Sanger sequencing is very laborious, time consuming and expensive.163 In the last decade next generation sequencing (NGS) techniques made it possible to investigate the DNA faster. With this novel technique a targeted region of the genome can be sequenced, such as a number of known disease genes, a certain (part of a) chromosome, or (almost) all coding parts, called whole exome sequencing (WES). Sequencing of the exons of known disease genes by using NGS was proven to be useful to detect the underlying pathogenic variant in heterogeneous diseases, such as retinopathies.164,165 WES, however, opens the possibility to identify

25 Chapter 1

A B C D

E F G

Figure 11: Several approaches to prioritize the variants identified by WES Examples of the approaches discussed in the text. The arrows indicate the persons in which WES is performed. Filled symbols in the pedigrees represent affected individuals, empty symbols represent presum- ably healthy individuals, and obligate carriers are depicted by a symbol with a dot. Circles below each pedigree symbolize the genetic variants identified, and the grey areas the most promising variants. A) Candidate region approach: disorder with a suspected X-linked inheritance, the shared variants between the probands on the X chromosome are the most promising candidates. B) Candidate region approach: disorder with a suspected autosomal dominant inheritance. The shared variants between the probands in the linkage area are the most promising candidates. C) Candidate region approach: consanguineous family with suspected autosomal recessive disorder in which the homozygous variants are the most promising candidates. D) Sib-pair approach: The overlapping variants following an autosomal recessive and X-linked inheritance are the most promising candidates. E) Phenotype first approach: variants in overlapping genes are the most promising candidates. F) Gene list approach: the variants in genes present on the list are the most promising candidates. F) De novo approach: the de novo variants and the variants following an autosomal recessive or X-linked inheritance are the most promising candidates. causal variants in genes not yet known to be associated with the disorder. Because many variants are identified by WES, several approaches are used to pinpoint the underlying causative variant, depending on the phenotype, the (expected) inheritance pattern, and molecular knowledge of the disorder.166 Five more commonly used approaches which have led to the identification of new associations between genes and disorders are depicted in Figure 11. Sometimes it is possible to define a candidate region, for example by using homozygos- ity mapping in consanguineous families or by using linkage analysis in large families with

26 Introduction multiple affected. The variants identified in the defined region can be further analyzed to find the underlying gene defect (Figure 11A-C).167-169 Secondly, in two affected sibs of non-consanguineous parents it is also possible to perform 170,171 sib-pair WES to identify the pathogenic variant (Figure 11D). In many patients, however, Chapter 1 there are no other affected family members. When there is a distinct clinical phenotype, sequencing of several probands can lead to the underlying gene defect, the phenotype first approach (Figure 11E).172-176 The fourth approach is to prioritize the variants by using molecular knowledge about the genes and mechanisms involved in the phenotype (Figure 11F). The application of gene lists based on the involved pathway or binding site of a specific transcription factor was success- ful in identifying the causal mutation.177,178 Because our molecular knowledge is far from complete, this strategy might lead to the exclusion of the pathogenic variant and therefore this approach is not often solely used. For CVI, however, none of these approaches are suitable: little is known about genes and pathways underlying CVI, the patients are often the only affected in the family, and the parents are mainly non-related. In addition, CVI is an umbrella diagnosis and therefore probably clinically and genetically heterogeneous, excluding a phenotype-based approach. In other heterogenic sporadic disorders, such as non-syndromic ID, epilepsy or autism, the trio approach was shown to be a useful strategy.179-182 By sequencing not only the patient but also the parents, de novo events can be detected and these de novo mutations appeared to be causal in 10-15% of the patients with a developmental disorder (Figure 11G).183,184 Moreover, this approach can also be used to search for pathogenic variants following an autosomal recessive or X-linked inheritance. In about 25% of individuals with developmental disorders a diagnosis can be established by using WES, and a possible diagnosis can be identified in up to 50% of the individuals.182-186 At the moment chromosomal investigations by using microarray is often performed before WES, but the methods to detect copy number variants, encompassing at least three exons, in WES data improve.187-189

Advantages to know the underlying pathology The knowledge about the underlying defect and a name for the disorder is already a relieve for parents. This allows the patient and/or parents to get in contact with other patients and parents with the same disorder and support each other on (non-)medical problems. Fur- thermore, information about the phenotypic spectrum and the long term prognosis can be beneficial for the patient. In disorders with a risk on CVI for example, early ophthalmological investigation and support can be given. Moreover, because of the critical period in visual development it is probably important to stimulate the vision as early as possible to achieve the best possible vision. Another question that often arises is whether there is a chance of

27 Chapter 1 recurrence; for themselves, a healthy brother or sister or other family members. Based on the inheritance pattern of the identified disorder a clear answer can be given. Furthermore, investigation of genes underlying CVI will improve our understanding of their function in the visual system and the molecular pathways involved, and this may give insight in the normal and pathological development and functioning of vision. Finally, this knowledge may lead to improved therapy, such as stimulation programs.

1.5 Scope, methods and outline of the thesis

The genetics underlying CVI has hardly been investigated. Therefore, we decided to study which genetic disorders were involved in the aetiology of CVI patients with low vision. We focussed on persons with low vision because of several reasons. First CVI can be difficult to diagnose. Therefore, we choose for patients with a quantifiable impairment: low visual acuity. Furthermore, CVI is a major cause of low vision in children, but the aetiology is only partly known.53 In addition, by including only patients with low vision a representative cohort (n=828) could be obtained, because it has been estimated that about half of all visually impaired children in the Netherlands are investigated in Bartiméus, institute for the visually impaired in Zeist, the Netherlands.53 The patient inclusion was based on the database in Bartiméus, in which the diagnosis and level of visual impairment was recorded by paediatric ophthalmologists.53 To search for patients with CVI the search terms listed in Box 1 were used, and only patients with low vision (VA ≤0.3 or <1.6 cycles/cm or visual field of ≤30°) were included.53

Box 1: Search terms Cerebral visual impairment Corpus callosum agenesia Cerebral anomaly Central nervous system anomaly unspecified Encephalopathy unspecified Motility disorder not leading to strabismus Neurological disorder unspecified Partial optic nerve atrophy

Of the 1144 patients obtained from this database (1975-2013) the medical files have been studied and patients with an additional major ocular disorder (e.g. cataract, retinitis pigmentosa, or retinopathy of prematurity) were excluded for further study. After applying these broad criteria 828 patients remained, which have been used for the studies in chapter 2 and chapter 3.

28 Introduction

1975 1985 1995 2005 2015

Chapter 2.1, n=309

Chapter 2.2, n=607 Chapter 1 Initial cohort, n=828

Figure 12: Overview of the patients selected for the different cohorts used in this thesis Years represent the first ophthalmological examination in Bartiméus of the patient included in the study.

As a proof of principle the medical files were studied for the presence of genetic disorders in patients with CVI with low vision (chapter 2). For the first study a recent 10-year period was used, because the awareness of genetic causes and the possibilities to diagnose genetic disorders has improved rapidly (chapter 2.1, Figure 12). The aetiology, co-morbidities and ophthalmological findings are reported for 309 CVI patients. In addition, it was investigated whether there were differences in the ophthalmological features between patients with an acquired cause versus those with a genetic aetiology. In chapter 2.2 the chromosomal aberrations identified in patients with CVI are evaluated in more detail. Because karyotyping is performed since 1970 a 20-year period was chosen to investigate the chromosomal aberrations.138 The aberrations identified were classified to investigate which chromosomal aberrations could lead to CVI.

For the studies described in chapter 3 and 4, we used more stringent criteria applied to the initial cohort of 828 patients. Based on the medical information in the files all patients with possible a risk factor for CVI (Box 2) or an identified genetic disorder were excluded.

Box 2: Possible risk factors for CVI Intrauterine CMV infection Maternal drug or alcohol abuse Perinatal problems Periventricular leukomalacia Brain haemorrhage Neonatal hypoglycemia Meningitis/encephalitis Severe brain trauma Hydrocephalus West syndrome

Subsequently, the selected patients and their parents were invited to participate in the study in Bartiméus (Figure 13). In one session the history was taken, and clinical examination was performed, which comprised the evaluation of the presence of dysmorphisms and ophthalmological investigation (Table 2). Blood was taken to perform DNA investigations, including targeted gene analyses, microarray and WES, in 56 patients and in most of the parents.

29 Chapter 1

Initial cohort (n=828) - Risk factor and/or known genetic disorder (n=631) - Deceased (n=58) Eligible (n=139)

- Declined (n=53) - Unreachable (n=30) Included for study described in chapter 3 and 4 (n=56)

Figure 13: Flow diagram of the patient inclusion for the study described in chapter 3

Table 2: Aspects of the ophthalmological examination Aspect Method used Fixation, eye movements and alignment Observation, little toys, covering of one eye VA TAC, LH, Landolt C, Snellen chart Visual fields Confrontative with Stycar balls or toy on a stick, Goldmann Anterior segment Slit lamp Retina, optic disc and fovea Funduscopy, OCT

In chapter 3.1 we investigate whether WES can be used to detect the underlying genetic defects in CVI. Moreover, this unbiased approach can lead to new associations between genes and CVI. Therefore, WES was performed in 25 patients and their parents. In chapter 3.2 the identification of compound heterozygous mutations in PGAP1 are discussed. PGAP1 is important in the GPI-anchoring pathway and was a candidate gene for intellectual disability. Further functional investigations were performed to strengtend the causal relation. In chapter 3.3 we report on the identification of heterozygous mutations in NR2F1 that lead to CVI, optic atrophy and intellectual disability. Whether the identified missense mutations lead to an aberrant protein function was investigated by a functional study. The overall results of the 56 CVI patients and a short discussion of these results are presented in chapter 4. In chapter 5 a general discussion is given. This chapter outlines the challenges in the interpretation of genetic variants in the light of CVI. Furthermore, the entity CVI and future perspectives are discussed.

Chapter 2

CVI: acquired and genetic causes

2.1

Low vision due to cerebral visual impairment: differentiating between acquired and genetic causes

Daniëlle G.M. Bosch, F. Nienke Boonstra, Michèl A.A.P. Willemsen, Frans P.M. Cremers, Bert B.A. de Vries.

BMC Ophthalmol 2014; 14: 59. Chapter 2.1

Abstract

Background To gain more insight into genetic causes of cerebral visual impairment (CVI) in children and to compare ophthalmological findings between genetic and acquired forms of CVI. Methods The clinical data of 309 individuals (mainly children) with CVI, and a visual acuity ≤0.3 were analysed for aetiology and ocular variables. A differentiation was made between acquired and genetic causes. However, in persons with West syndrome or hydrocephalus, it might be impossible to unravel whether CVI is caused by the seizure disorder or increased intracranial pressure or by the underlying disorder (that in itself can be acquired or genetic). In two subgroups, individuals with ‘purely’ acquired CVI and with ‘purely’ genetic CVI, the ocular variables (such as strabismus, pale optic disc and visual field defects) were compared. Results It was possible to identify a putative cause for CVI in 60% (184/309) of the cohort. In the remaining 40% the aetiology could not be determined. A ‘purely’ acquired cause was identified in 80 of the patients (26%). West syndrome and/or hydrocephalus was identified in 21 patients (7%), and in 17 patients (6%) both an acquired cause and West syndrome and/ or hydrocephalus was present. In 66 patients (21%) a genetic diagnosis was obtained, of which 38 (12%) had other possible risk factor (acquired, preterm birth, West syndrome or hydrocephalus), making differentiation between acquired and genetic not possible. In the remaining 28 patients (9%) a ‘purely’ genetic cause was identified. CVI was identified for the first time in several genetic syndromes, such as ATR-X, Mowat- Wilson, and Pitt Hopkins syndrome. In the subgroup with ‘purely’ acquired causes (N=80) strabismus (88% versus 64%), pale optic discs (65% versus 27%) and visual field defects (72% versus 30%) could be observed more frequent than in the subgroup with ‘purely’ genetic disorders (N=28). Conclusions We conclude that CVI can be part of a genetic syndrome and that abnormal ocular findings are present more frequently in acquired forms of CVI.

36 Low vision due to CVI: differentiating between acquired and genetic causes

Background

Cerebral visual impairment (CVI) is one of the major causes of low vision in the developed world and accounts for 27% of the visually impaired children.53 Visual impairment in CVI is due to a disorder in projection and/or interpretation of the visual input in the brain.47 In the absence of absolute criteria for the diagnosis, the following definition is commonly used: CVI includes all visual dysfunctions caused by damage to, or malfunctioning of, the retrochiasmatic pathways in the absence of any major ocular disease.51 A more practical definition Chapter 2.1 includes an impairment of vision with normal function of the ocular structures and anterior visual pathways.62,79,119 The main features of CVI during ophthalmological investigation are: impaired visual acuity, visual field defects, and abnormal visual behaviour, while obvious ocular abnormalities are not found. This visual behaviour consists of looking away from the target while reaching to it, looking past the target, staring into lights, and fluctuating visual performances. Fixation abnormalities can be observed, such as a prolonged time before fixat- ing on a stimulus and intermittent fixation towards a stimulus.58,190 The visual acuity can range from blindness to normal, and is in CVI due to perinatal causes correlated to the severity of the brain damage.50,70,72 Crowding, the impairment of the ability to recognize objects in clutter, is present in more than 40% of the normally sighted CVI individuals, however, in individuals with CVI and low vision the percentage of crowding is unknown.41,72,74 The occurrence of strabismus (37-73%) and nystagmus (12-73%) varies highly among previous reports.51,79,119 In funduscopy a (segmental) pale optic disc can be seen (25-44%), whereas in premature born individuals a small optic disc or a more pronounced excavation can be found.112 Remarkably, the latter could also be observed in individuals with mutations in NR2F1, which were recently identified.191 Visual fields can be impaired, varying from partial visual field defects, to severe constriction of visual fields.77 It has been demonstrated that visual evoked responses do not differ between individuals with and without CVI, although the responses can be abnormal.192 In addition, fixation is often severely impaired in CVI and minimal fixation is necessary for visual evoked responses. Therefore, visual evoked responses cannot be used to diagnose CVI. Furthermore, difficulties with object or face recognition and visiospatial disorders can be observed.46,58 Neuropsychological testing can be helpful to gain more insight in specific higher perceptual deficits in the individual, but are only applicable to children with a developmental age above two years and 9 months.85 For a first screening of higher perceptual functions several questionnaires have been developed.72,83 In general, the causes of CVI can be divided into acquired and genetic forms. The acquired forms can be derived prenatally (e.g. intrauterine infections), perinatally (e.g. ischaemic brain injury), and postnatally (e.g. hypoglycemia or meningitis). Perinatal problems, often due to premature birth, are the most frequent causes of acquired CVI, occurring in up to two-thirds of the cases, whereas neonatal meningitis and/or encephalitis has been reported as important causes in the postnatal period (2.5-12%).47,51,79,112,119 Other acquired causes of CVI, such as

37 Chapter 2.1 head trauma and brain tumours are less common.51,62,79 In complex cases, like children with severe epilepsy or hydrocephalus, it might be impossible to unravel whether CVI is caused by the seizure disorder or increased intracranial pressure or by the underlying disorder (that in itself can be acquired or genetic).115,116 CVI, either acquired or genetic, is often part of a more complex phenotype, and co-occurrence of non-ophthalmic impairments and disorders, such as intellectual disability or epilepsy, are common.51,79 Knowledge about the occurrence of CVI in persons with or without intellectual disability can alter the way of approaching the person. For example, it is important to offer objects one by one or for a longer period of time, in case of crowding or fixation abnormalities. Moreover, rehabilitation of children with CVI can improve their overall function.193 Although few studies reported on large series of 100 to 170 individuals with CVI, little attention is paid to whether genetic disorders are involved.51,79,119 Several studies have been performed on higher perceptual deficits in more common genetic syndromes, such as Williams syndrome and Prader-Willi syndrome.121,194 However, in the description of these patients in those reports a reduced visual acuity is not mentioned. Our goal was to get more insight into the genetic causes of CVI with low vision. Because in acquired forms of CVI the mechanism and the moment of disruption are different compared to CVI due to genetic causes, we also aimed to investigate whether different ocular phenotypes could be observed for genetic versus acquired forms.

Methods

All individuals with CVI included in this study were seen in Bartiméus, an institute for diagnostics, rehabilitation and schooling of the visually impaired in the Netherlands, in the period 2002-2012. Assuming that the incidence of visual impairment in 3.1 million Dutch children aged 0–15 years is similar to the 8 / 100 000 per year reported in Scandinavia, approximately half of the annually recorded children with low vision in the Netherlands are seen at Bar- timéus.53 All children were referred to Bartiméus by medical specialists, i.e. paediatricians, rehabilitation physicians, ophthalmologists, general physicians and orthoptists. The children were referred for different reasons, mostly because of suspected visual impairment. A minority was referred for further diagnosis such as electrophysiology. The majority of children lived at home with their parents and were seen in the outpatient facilities of the Institute. All investigations were performed by a paediatric ophthalmologist with assistance of a technical ophthalmological assistant and/or orthoptist. Visual acuity was measured mono- and bin- ocularly with correction of the refraction error under controlled light conditions. Monocular vision was measured by covering one eye by a special device for occlusion which could be added to the glasses worn by the patient. Visual acuity in young children or in individuals with a young developmental age was measured by forced preferential looking (Teller Acuity

38 Low vision due to CVI: differentiating between acquired and genetic causes

Cards, 55 cm), or with “LH Symbols” (3 m).66 The confrontational method with white Stycar balls, 5 cm, was used to estimate the visual fields. The balls on a stick were presented in all quadrants by a person behind the individual investigated. A person in front looked for response to the presented object: eye movements, pointing or a verbal response. Eye alignment, fixation, following, and visual behaviour were observed. A handheld slit lamp was used to assess the anterior segment. In mydriasis, funduscopy and retinoscopy were performed, whenever the patient and parents agreed. But in case of lack of cooperation funduscopy and retinoscopy were not always possible. Electroretinography was rarely performed, because of Chapter 2.1 developmental age or behavioural problems. CVI was diagnosed when there was no other ocular diagnosis which could explain the visual impairment or visual field defect, and/or typical features such as poor fixation or crowding were present, and/or CVI was found at neuropsychological investigation. Neuropsychological investigation of the visual functions was, however, not possible in a majority of the individuals, because of their developmental age. Although, there are tests available from 2+9 years onwards, the tests used in Bartiméus in the past 10 years were only applicable in patients with a developmental age above six years. Crowding was measured with the C-test at 5m or with a LH Symbols version of the C-test on 40 cm as described in Huurneman et al. and was defined increased when the crowded ratio was ≥2.67,74,195 Additional inclusion criteria were a first visit over a 10 years period between 1-1-2002 and 1-1-2012 and low vision, defined as a visual acuity of ≤0.3 or <1.6 cycles/cm at 55 cm or a visual field radius of ≤30° degrees.53 Under the age of three the visual acuity was measured by Teller Acuity Cards and defined as decreased, when the acuity in cycles/degree was below the normal range for their age reported by Courage and Adams.196 Exclusion criterion was a second ocular diagnosis causing low vision, such as cataract or retinopathy of prematurity. Optic nerve atrophy was not an exclusion criteria, because it can occur as a result of retrograde transsynaptic degeneration in CVI.105 Primary (hereditary) optic atrophy or congenital idiopathic nystagmus were excluded based on the history and results during ocular examination by the ophthalmologist. Bilateral amblyopia was excluded as refraction was measured and corrected if necessary. The most recent ophthalmologic examination was used for further analyses, including binocular visual acuity, visual fields, strabismus, nystagmus, refraction error and the aspect of the optic disc. The use of vigabatrin was registered because of the risk of constriction of visual field.197 To identify potential causes of CVI we evaluated the genetic investigations, risk factors during pregnancy, birth, and neonatal/childhood period, and reports on cerebral imaging. For the comparison of the ophthalmological findings the data of the individuals with acquired causes and with a genetic diagnosis were used. To avoid confounding factors, which may contribute to CVI, additional criteria were used to select the subgroups for this comparison. Individuals with West syndrome and hydrocephalus as well as individuals with a genetic diagnosis in combination with a gestational age <37 weeks, unknown gestational

39 Chapter 2.1 age or acquired causes (e.g. perinatal problems) were excluded. For the statistical analysis the Mann-Whitney U test and the Fisher’s Exact test were used. A p-value below 0.05 was considered to be significant. To control for the false discovery rate at 0.05 we used the Benjamini-Hochberg method.198 This study was approved by the Ethics Committee of the Radboud university medical center (Commissie Mensgebonden Onderzoek, regio Arnhem- Nijmegen). According to the Committee informed consent was not necessary, because all data were processed anonymously. Local approval of the institute was obtained. The study was conducted according to the tenets of the Declaration of Helsinki.

Results

309 individuals with low vision fulfilled the criteria for CVI. The general information about the cohort is presented in Table 1 and in more detail in Additional file 1. Almost half of the patients (151/309) had their first ophthalmological examination before the age of three years, whereas five individuals were over 18 years of age. 152 persons had more than one examination, and there was a median interval of 28 months between the first and the most recent examination. The persons without a second examination (n=157) had a median age of 46 months (5 months -45 years). In 20% (62/309) of the cohort there was a vision below 0.05 or 1.6 cycles/cm at 55 cm (Teller acuity cards) (Table 2). High refraction errors, myopia <-4 or hypermetropia >+4, were present in 25% (42/172) of the persons. In 77% (226/295) a strabismus and in 42% (113/270) a nystagmus was present. Visual field defects were found in 60% (149/249), consisting of constricted visual fields in half of the individuals. (Partial) pale optic discs were present in 44% (128/293). Fixation problems were present in 45% and fluctuating visual performances 31%. Co-morbidities were found in a high percentage, such as intellectual disability (96%, 298/309, IQ<70 or developmental delay) and hearing impairment (12%, 24/196, hearing threshold above 25 dB) (ICD-10, http://www.who.int/classifications/icd/en/) (Table 1). In 32% (71/221) the gestational age was below 37 weeks, of which two-thirds were born before 35 weeks. In 67% of the cohort a MRI-scan was performed, which was reported normal in only 14% (28/206). Brain anomalies were either caused by a disturbance in the developmental process (e.g. lissencephaly, cortical dysplasia) and white matter disease (e.g. hypomyelination) or acquired (e.g. periventricular leukomalacia (PVL), stroke). We subdivided the patients in a normal MRI group and an abnormal MRI group and searched for the difference in ophthalmologic features. In the patients with an abnormal MRI scan more pale optic disc were seen (49% versus 14%, p=0.001) (Additional file 2). It was possible to identify one or more possible causes for CVI in 60% of the cohort. In 23% there was evidence of severe perinatal problems, such as haemorrhage or infarctions, PVL or post-partum reanimation. In 11% exogenous factors led to brain damage and CVI,

40 Low vision due to CVI: differentiating between acquired and genetic causes

Table 1: General information, co-morbidities, aetiology of the cohort General information Median (range) Age first examination (months) 36 (4 months- 45 years) Age most recent examination (months) 53 (5 months – 45 years) Birth weight (gram) 2980 (760-4750) Gestational age (weeks) 39 (27-42) Regarding N/ Available N (%) Men 170/309 (55%)

Mortality 15/309 (5%) Chapter 2.1 Twins 20/309 (6%) Gestational age <37 weeks 71/222 (32%) Vigabatrin use 44/309 (14%) Anomalies MRI-cerebrum 178/206 (86%) Co-morbidities Affected N/ Available N (%) Intellectual disability 298/309 (96%) Motor impairment 288/294 (98%) Hearing impairment 24/196 (12%) Aetiology Number (%) Perinatal problems* 70 (23%) PVL* 35 Stroke* 32 Exogenic* 33 (11%) Meningitis/encephalitis * 13 Congenital CMV infection* 6 Head trauma* 6 Complication of operation/co-morbidity* 8 Genetic diagnosis* 66 (21%) Hydrocephalus* 20 (6%) West syndrome* 28 (9%) Unknown 125 (40%) *Can coexist.

such as meningitis or encephalitis, a diabetic coma, or a complication during an operation. Hydrocephalus was present in 6% and West syndrome in 9% of the cohort. A genetic diagnosis was reported in 21% (Table 3), of which 50% had a proven chromosomal aberration. In half of the persons with a genetic syndrome other factors, such as West syndrome or stroke, may have contributed to the CVI. In the other half the CVI might be associated with the genetic syndrome. After excluding individuals who had multiple factors that could have contributed to CVI a subgroup (N=108) remained, which allowed for the comparison of the ocular findings be-

41 Chapter 2.1

Table 2: Findings of ophthalmological examinations Ocular findings Affected N/ Available N (%) Myopia < -4 13/171 (8%) Hypermetropia > +4 29/171 (17%) Visual acuity < 0.05 or <1.6 cycles/cm at 55 cm 62/309 (20%) Strabismus 226/295 (76%) Nystagmus 113/270 (42%) Eye movement disorders 100/283 (35%) Visual field defect 149/249 (60%) Hemianopia 28/149 (19%) Upper or lower visual field defect 37/149 (25%) Constriction of visual field 84/149 (56%) (Partial) pale optic disc 128/293 (44%) Visual behaviour/ Percentage higher visual disorders Fluctuating visual performances 31% Fixation abnormalities 45% Looking away from the target 19% Crowding 8% Problems with object/face recognition 7% Staring at lights 15% Auditive stimuli dominates 9%

tween individuals with a ‘purely’ acquired form of CVI (N=80) and those with a ‘purely’ genetic form (N=28). There were no significant differences between the groups according to the age at the ophthalmological examination (72 months versus 54 months, p=0.333) or the use of vigabatrin (9% versus 7%, p=1.00), of which a known side effect is visual field constriction defects (Table 4).197 The occurrence of blindness (16% versus 25%, p=0.396), nystagmus (46% versus 35%, p=0.357) or high refraction errors (myopia: 4% versus 0%, p=0.567; hypermetropia; 6% versus 11%, p=0.425) did not differ significantly between the groups. In the ‘purely’ acquired group, however, strabismus (88% versus 64%, p=0.009), visual field defects (72% versus 30%, p=0.001) and (partial) pale optic discs (65% versus 27%, p=0.001) were observed more often than in the ‘purely’ genetic group. The type of visual field defects, concentric, hemianopia, or upper or lower visual field defects, did not seem to differ between the two groups, but the numbers were too small to make definite conclusions.

42 Low vision due to CVI: differentiating between acquired and genetic causes

Table 3: Syndromic diagnosis in patients with CVI ID Diagnosis* GA Contributing Age at CVI Evidence Gene factors examination previously for genetic (months) reported diagnosis 269 Aicardi syndrome 36 W 50 199 C 316 Aicardi syndrome 42 W 108 C 173 Aromatic decarboxylase deficiency 32 38 200 C,M 319 ATR-X 38 70 C,G ATRX

55 CDG type 1a 41 22 126,127 C,M

305 CDG type 1a NA 25 C,M Chapter 2.1 223 Citrullinaemia NA 81 C,M 128 Coffin-Siris syndrome 41 84 C 43 Cohen syndrome 38 53 C

433 Complex I deficiency NA 15 201,202 C,M 378** Complex I deficiency 40 35 C,M 403 Complex II deficiency 41 46 C,M

5 Complex III deficiency 41 W 73 202 C,M 266 Complex I and III deficiency NA 50 C,M 29 Complex II and III deficiency 42 S 48 C,M 175 D2-hydroxyglutaaraciduria NA 20 203-205 C,M 425 Incontinentia Pigmenti 40 S, W 67 206,207 C 184 Infantile neuroaxonal dystrophy 39 45 208,209 C 325 Copper storage disorder 41 27 210 C,M 263 Lissencephaly NA 38 211 C,G DCX 231 Marden-Walker syndrome 35 79 C 17 Mowat-Wilson syndrome 41 34 C,G ZEB2 215 Opitz C syndrome NA 199 C 216 Pelizaeus-Merzbacher syndrome 38 27 62 C,G PLP1 192 Pitt-Hopkins syndrome 40 35 C,G TCF4 Pontocerebellar hypoplasia type 2 C 85 36 30 212 Triple X C,G 238 Propion acidemia 36 117 213 C,M 78 Rett syndrome 36 23 C,G MECP2

99 Rett syndrome 38 20 214 C,G CDKL5 172 Rett syndrome NA 14 C,G CDKL5 364 Rett syndrome NA 197 215 216 C 341 Tubereus sclerosis NA Compression 179 C

from tubers 62 41 Tubereus sclerosis NA Compression 165 C from tubers 348 Vanishing white matter NA 28 217 C *in an additional 33 individuals chromosomal aberrations were identified. **previously described in Mora- va et al.218 ATR-X syndrome= Alpha-Thalassemia X-linked intellectual disability syndrome, CDG = congenital disorder of glycosylation, C= clinical diagnosis, GA= gestational age, G=gene mutation, M= metabolic diagnosis, NA= not available, P= perinatal problems, S= stroke, W= West syndrome. The syndromes in the bold formatted rows are for the first time associated with CVI.

43 Chapter 2.1

Table 4: Ocular findings in individuals with a ‘purely acquired’ or a ‘purely genetic’ cause Acquired (N=80) Genetic (N=28) Raw p-value Mean age most recent examination 70 (SD 58) 54 (SD 39) 0.333 (months) Affected N/ Available N Affected N/ Available N Men 48/80 (60%) 17/28 (61%) 1.00 Vigabatrin use 7/80 (9%) 2/28 (7%) 1.00 Abnormal MRI 55/56 (98%) 11/14 (79%) 0.023 Myopia <-4 3/80 (4%) 0/28 (0%) 0.567 Hypermetropia >+4 5/80 (6%) 3/28 (11%) 0.425 Visual acuity <0.05 or <1.6 cycles/cm 13/80 (16%) 7/28 (25%) 0.396 at 55 cm Strabismus 68/77 (88%) 18/28 (64%) 0.009* Nystagmus 31/67 (46%) 9/26 (35%) 0.357 Visual field defect 47/65 (72%) 7/23 (30%) 0.001* Hemianopia 10/47 1/7 - Upper or lower visual field defect 19/47 3/7 - Constriction of visual field 18/47 3/7 - (Partial) pale optic disc 51/78 (65%) 7/26 (27%) 0.001* *p-Values represent differences that passed the Benjamini–Hochberg criterion (false discovery rate at 0.05).

Discussion

We analysed causes of CVI in a large cohort of 309 individuals with low vision in the Nether- lands. Although CVI has also been described in individuals with a (sub-)normal visual acuity and higher perceptual deficits, within this study only patients were selected with a vision ≤0.3 or a visual field below 30 degrees.46,50,72 For the purpose of this study, we excluded persons with major anterior ocular diseases, although CVI can also be present in those patients. In some persons only grating acuity (Teller Acuity Cards) could be measured. In general using grating acuity initially may lead to higher scores than the optotype acuity that can be applied later in development.219 Although some children were investigated at a very young age, which might hamper the differentiation between CVI and delayed visual maturation (DVM), about half of the individuals had more than one ophthalmological examination, with a median interval of 28 months between the first and the most recent examination, whereas another quart of the persons were over 48 months allowing for an accurate clinical diagnosis. We hypothesized that in children with DVM there is a concordant delay in visual functions. In CVI, on the contrary, there may be discrepancies in the developmental stage of different visual functions. When this hypothesis is correct, it might be possible to differentiate earlier between CVI and DVM by signalling discrepancies in the profile of visual functions.

44 Low vision due to CVI: differentiating between acquired and genetic causes

This study was based on the medical files, in which sometimes information is missing, for example on ERG, ophthalmological results or MRI scan. In addition, for some features (e.g. fixation) only abnormalities were reported, although this was probably assessed in all patients but not always recorded. However, we were able to collect a large group of patients and in the majority of these patients extended investigations were performed. The ocular findings in our cohort were comparable to previous reports, except for the visual field defects, of which a higher percentage was found in our study (60% versus 9%).51,79 In two-thirds of the patients an MRI scan was performed. In only 14% of the individuals with an MRI scan Chapter 2.1 of the brain, the images were reported to be normal (28/206), which is comparable with a previous study by Khetpal et al.79 The spectrum of MRI abnormalities in our cohort was broad: from structural abnormalities, such as corpus callosum dysplasia, holoprosencephaly or lissencephaly, to white matter abnormalities, such as hypomyelination and PVL. This shows that many different mechanism that lead to brain disease may cause CVI. In individuals with a normal MRI scan or only a thin corpus callosum, the pathogenesis was less clear. Pale optic discs were seen more frequently in the patients with reported anomalies on MRI scan. However, as the MRI scans were assessed by many radiologists with different expertise we are reluctant to draw firm conclusions. In 60% of the individuals, we were able to identify the cause of the CVI, of which 32 persons were classified in more than one group. In 34% evidence of acquired problems, mainly perinatal, were found which is lower than the 42% - 67% previously reported.51,79,119 This might be due to more stringent criteria used in our study, as only individuals with known brain damage on cerebral imaging, twin-twin transfusion syndrome or postpartum resuscitation were classified as perinatal problems. In 40% of the patients we could not identify a cause for CVI. This can be due to missing information in the medical file, a not recognized acquired cause (for example an unrecognized CMV infection during pregnancy), a not identified genetic cause or a combination of several minor acquired and genetic risk factors. In 21% of the individuals a genetic diagnosis was obtained, of which 50% had proven chromosomal aberrations (Table 3). As the techniques to identify underlying genetic causes are improving rapidly, this 21% is probably an underestimation. We observed various genetic syndromes for which an association with CVI has previously been reported: trisomy 21, congenital disorder of glycosylation (CDG) type 1A, complex II deficiency, copper storage diseases, infantile neuroaxonal dystrophy, Pelizaeus-Merzbacher syndrome and Rett syndrome.62,126,127,130-132,202,208-210,214-216 However, for the following genetic syndromes such association was not reported so far: Alpha-Thalassemia X-linked intellectual disability syndrome, Coffin-Siris syndrome, Pitt-Hopkins syndrome, Mowat-Wilson syndrome and Cohen syndrome. In some of these syndromes retinal diseases have been described, for example in Cohen syndrome or CDG type 1A.126,220 In our cohort an electroretinography was often not available.

45 Chapter 2.1

However, as funduscopy did not reveal any retinal abnormalities, retinopathy was unlikely to cause visual impairment. In neurodegenerative diseases, such as complex II deficiency, optic atrophy can occur as a result of mitochondrial dysfunction, but these disorders affect the brain equally.202 Moreover, retrograde transsynaptic atrophy may occur, making differentiation between CVI and visual impairment due to optic atrophy in these disorders difficult. Although CVI might still be a coincidental finding in the above mentioned genetic syndromes, most individuals with these or other syndromes have not systematically been tested for CVI. For example, visual acuity, visual behaviour, and the findings at funduscopy are sel- dom reported in the clinical reports and children with an intellectual disability are not always seen by a paediatric ophthalmologist. Therefore, it is likely that CVI might be a consistent part of these syndromes, but a thorough assessment of the visual functions in more individuals with these syndromes is necessary to establish this association. We were able to establish a diagnosis in the majority of CVI in our study population, consisting of genetic disorders and acquired causes. After exclusion of possible confounders a ‘purely’ genetic and a ‘purely’ acquired subgroup remained. Between those two different aetiologies the ophthalmological examination revealed significant differences (Table 4). Strabismus, visual field defects and (partial) pale optic disc were significantly less common in the genetic group compared to the acquired group. This might be explained by the localiza- tion of the defect as in the acquired group, the most important cerebral damage was PVL and stroke, both mainly localized in the optic radiations. Damage to the optic radiations can cause visual field defects and retrograde optic disc pallor.61,105,112 In the genetic group the optic radiations are probably less severely affected and a more overall dysfunction of the brain and/or cortical anomalies might have resulted in the visual impairment. This theory is supported by the observation that normal optic discs are seen more often in individuals with cortical lesions compared to subcortical lesions.61 A comparable mechanism might result in the higher frequency of strabismus in the acquired group. To guide the alignment of the visual axes the binocular connections in the striate cortex play an important role.221 When the input to the striate cortex is disturbed by prism-glasses, strabismus develops in primates.222 So it can be expected that injury to the optic radiations by stroke or PVL, which also disrupts the input to the striate cortex, leads to strabismus. Although there are more mechanisms that cause strabismus, the above mentioned probably accounts for the difference between the two groups.

Conclusions

In conclusion, we presented a large cohort of mainly children with CVI and impaired vision. Genetic disorders were present in 21% of patients and an association between the disorder and CVI is considered. CVI patients with genetic disorders showed an overall different and

46 Low vision due to CVI: differentiating between acquired and genetic causes milder ocular phenotype: less frequently strabismus, visual field defects and pale optic discs. Moreover, we observed CVI for the first time in several known genetic syndromes making the screening of visual functions in those syndromes recommendable, to ensure the best approach and clinical care to the often multiple disabled persons. Also clinical genetic investigation should be considered in persons with CVI without evidence of an acquired cause. Awareness of CVI as part of a genetic disorder is currently increasing, and the techniques to identify the underlying genetic defect are improving rapidly. Therefore, it is likely that more genetic defects will be identified to be associated with CVI in the near future. Chapter 2.1

47 Additional files Vigabatrin Yes Yes Yes Yes I cerebrum abnormalities M R I cerebrum (based on reports) Normal Hyperdense abnormalitiesHyperdense dorsal thalamus Corpus callosum agenesis Corpus NR Normal Normal Atrophy of cerebellar hemispheres, hemispheres, of cerebellar Atrophy peduncles and vermis. NR Thin corpus callosum, corpus Thin matter white periventricular abnormalities Brain dysgenesis Brain NR Small corpus callosum, agenesis Small corpus genu and rostrum Hypomyelination of the central of the central Hypomyelination and occipital matter white Hypomyelination isk R factor W W PVL, S W W Genetic diagnosis Deletion 2q Complex III Complex deficiency Mowat-Wilson Mowat-Wilson syndrome Classification Unknown Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Genetic Unknown ‘Purely’ acquired ‘Purely’ Genetic Unknown Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Unknown Unknown Unknown ‘Purely’ genetic ‘Purely’ earing H impairment ------Developmental Developmental calender age at age 6 months at 6 at 6 months years 3-4 months at at 3-4 months 1+10 years 11-15 months 11-15 months 42 months at IQ 55 9 months at 30 at 9 months months 8 months at at 8 months 1+4 years Intellectual disability + + + + + + + + + + + + + + Gestational Gestational age 39 39 40 40 41 41 38 40 38 39 38 41 40 41 Birth weight (gram) 3720 3480 3200 2750 4490 3415 4350 2840 3500 2600 3525 3855 3105 3810 emale Male/ F Male Male Female Male Male Female Female Female Female Male Female Female Female Male dditional file 1: Phenotype information of all patients involved, part 1 involved, of all patients dditional file 1: Phenotype information 2 ID 1 4 3 7 5 6 9 12 11 15 16 18 17 A

48 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) Periventricular leukomalacia Periventricular parieto-occipital, hydrocephalus Hypomyelination Hypoplasia and delayed Hypoplasia and delayed callosum of corpus myelination Major intraventriculair bleedings Major intraventriculair Almost complete agenesis of complete Almost vermis. Supratentorial central atrophy and atrophy central Supratentorial atrophy. frontotemporal peripheral ventricles. Enlarged Mega cisterna magna, corpus corpus Mega magna, cisterna colpocephaly, callosum agenesis, delayed atrophy, cerebral myelination of cerebellar absence Almost remainings Possible hemispheres. Possible of haemorrhages. matter white temporal anterior abnormalities NR Normal isk R factor H, PVL, S W H, S S Genetic diagnosis Complex II and III Complex deficiency Classification Unknown Acquired Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Unknown Unknown Acquired Genetic and acquired Unknown Unknown earing H impairment - - - - - Developmental Developmental calender age at age 28 months at at 28 months 42 months 2 years at 2+6 at 2 years years 6 months at 9 at 6 months months 9-11 months at at 9-11 months 2+11 years Intellectual disability + + + + + + + + + + Gestational Gestational age 36 40 41 41 40 39 42 38 Birth weight (gram) 2460 3725 4210 3605 3610 2915 3075 4275 2820 emale Male/ F Female Male Female Female Male Male Female Female Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information ID 19 22 20 25 28 27 32 29 31 33 A

49 Chapter 2.1 Vigabatrin Yes Normal I cerebrum abnormalities M R I cerebrum (based on reports) Diffuse diminished brainvolume. Diffuse diminished brainvolume. mainly Gliosis along ventricles, occipital. Small cerebellum lobe and temporal Polymicrogyria, abnormal fissure along Sylvian matter of white signal Delayed myelination. Delayed NR Delayed myelinisation, enlarged enlarged myelinisation, Delayed mega small cerebellum, ventricles, magna. cisterna Intracerebral tubers Intracerebral Wide ventricles, thin corpus thin corpus ventricles, Wide callosum, thin pituitary stalk Delayed myelination, myelination, Delayed leukomalacia periventricular Wide periphere liquor areas periphere Wide NR isk R factor COM PVL Deletion 6q15-q16.1 Genetic diagnosis Deletion 6p25.3- Duplication pter, 20q13.33-qter Cohen syndrome Cohen Tubereus Tubereus sclerosis Duplication 4q32.2 Unknown Genetic Classification Unknown Unknown Genetic Unknown Unknown ‘Purely’ genetic ‘Purely’ Genetic and acquired genetic ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ earing - H impairment ------+ - - Developmental Developmental calender age at age 1+4 years at at 1+4 years 3+2 years 15-17 months 2+9 years at 11 months at at 11 months 2+6 years 4 years at 10 at 4 years years 9-10 months at at 9-10 months 22 months 1,5-3 months at at 1,5-3 months 33 months Intellectual disability + + + + + + + + + + + + Gestational Gestational age 41 42 41 40 38 37 38 34 Birth weight (gram) 3980 2800 4110 4000 2710 3800 2025 emale Female Male/ F Male Female Female Male Male Female Male Male Male Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 36 37 ID 34 46 47 39 45 43 42 40 48 41 A

50 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Chapter 2.1 Tissue loss, asymmetric ventricle asymmetric ventricle loss, Tissue system I cerebrum abnormalities M R I cerebrum (based on reports) Abnormal Small cerebellum and vermis, and vermis, Small cerebellum myelination. delayed Periventricular leukomalacia Periventricular Periventricular leukomalacia Periventricular Mild matter white periventricular cerebellar dysplasia abnormalities, vermis NR Enlarged ventricles and ventricles Enlarged leukomalacia. periventricular Cortical with loss of necrosis cortical and subcortical and white Remainings of old matter. gray bleeding with gliosis. Periventricular leukomalacia, hardly hardly leukomalacia, Periventricular myelination any Normal NR Periventricular leukomalacia Periventricular Much liquor, twisted optic nerves Much liquor, NR isk R factor S P PVL PVL, S PVL S, T S, PVL PVL Genetic diagnosis Trisomy 21 Trisomy CDG type 1a Trisomy 21 Trisomy ‘Purely’ acquired ‘Purely’ Unknown Classification Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ Genetic ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ Genetic Unknown ‘Purely’ acquired ‘Purely’ earing - H impairment ------Developmental Developmental calender age at age 5-8months at at 5-8months 2+8 years IQ 72 6-10 months at at 6-10 months 3 years IQ 76 + Intellectual disability + + + + - + + - + + + + - + Gestational Gestational age 41 40 31 35 27 37 39 32 29 31 Birth weight (gram) 3290 3460 1510 2050 1090 3186 2190 1355 1000 1950 emale Male Male/ F Female Male Male Male Male Male Male Female Male Male Female Female Male Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 51 55 54 50 ID 49 67 68 70 66 64 57 56 73 72 74 A

51 Chapter 2.1 Vigabatrin Yes Yes I cerebrum abnormalities M R I cerebrum (based on reports) NR NR NR Congenital media stroke left. media stroke Congenital Deep tissue. of cerebral Necrosis and basal ganglia matter white high signal. showed Thin corpus callosum, immature callosum, immature corpus Thin brain NR NR Pontocerebellar hypoplasia type 2 hypoplasia Pontocerebellar Abnormalities frontal and in the Abnormalities frontal basal ganglia, thalamus. White matter abnormalities matter White periventricular Normal NR Delayed myelination Delayed isk R factor W S I PVL H P, W P, W Genetic diagnosis Pontocerebellar Pontocerebellar type 2 hypoplasia Rett syndrome Unknown Unknown Classification Unknown Unknown Hydrocephalus and/ Hydrocephalus syndrome West or Unknown ‘Purely’ acquired ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Genetic Acquired Genetic Hydrocephalus and/ Hydrocephalus syndrome West or earing - - H impairment - - - + ------Developmental Developmental calender age at age 23-26 months 23-26 months 2+10 years at 4,5 months at at 4,5 months 19 months Intellectual disability + + + + + + + + + - + + + + Gestational Gestational age 36 36 37 37 33 36 36 39 36 Birth weight (gram) 1900 2750 2480 3500 1810 3030 2710 emale Male/ F Male Female Female Male Female Male Female Female Male Female Female Male Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 83 81 77 ID 75 93 89 92 88 87 85 84 79 78 95 A

52 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Yes Yes Chapter 2.1 NR I cerebrum abnormalities M R I cerebrum (based on reports) Multicystic encephalopathy, Multicystic encephalopathy, venticles. and lateral third enlarged Ischaemic damage thalamus and left damage due to occipital abcess. NR Multicystic encephalopathy with Multicystic encephalopathy dilatation ventricle Hypoplasia of the frontotemporal Hypoplasia of the frontotemporal fossa till the posterior areas NR Asymmetric dilatation of ventricles. of ventricles. dilatation Asymmetric matter white Periventricular abnormalities with punctate delayed Slightly hypointensity. myelination. Small choroid plexus cyst plexus Small choroid NR isk R factor COM I I,S PVL PVL, S Genetic diagnosis Deletion 21q22.13-q22.3, Duplication 18p11.32-p11.21 Duplication Duplication 11q23.3-qter, Deletion 12q24.3-qter Rett syndrome Trisomy 21 Trisomy Deletion 5q35 ‘Purely’ acquired ‘Purely’ Classification ‘Purely’ acquired ‘Purely’ Unknown Genetic and acquired ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ Genetic ‘Purely’ genetic ‘Purely’ earing - H impairment ------Developmental Developmental calender age at age 20 months at at 20 months 33 months 14 months at 2 at 14 months years 9 months at at 9 months 2+3 years Intellectual disability + + + + + + + + + + Gestational Gestational age 42 38 39 38 40 41 31 40 3500 Birth weight (gram) 3640 3515 3760 1590 2170 emale Female Male/ F Female Female Male Female Male Female Male Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 97 99 ID 96 104 103 102 101 108 107 105 A

53 Chapter 2.1 Vigabatrin Yes I cerebrum abnormalities M R I cerebrum (based on reports) NR Cortical dysplasia frontal leftCortical frontal dysplasia NR Progressive cortical degeneration. Progressive hemorhages Intracerebral NR Dysgenesia callosum and of corpus cyst intraventricular myelination Incomplete NR Cystic abnormalities, mainly in abnormalities, Cystic and left basal fossa, posterior ganglia. Gliosis periventricular Gyration disorder along the Sylvian along the Sylvian disorder Gyration fissure NR atrophy, cerebral liquor areas, Wide small corpus matter, little white callosum. Corpus callosum agenesis with Corpus colpocephaly Periventricular leukomalacia Periventricular NR isk R factor H, I S, T S, S P I PVL S, T S, PVL Genetic diagnosis Deletion 15 Duplication 14 Duplication and 8 Coffin-Siris Coffin-Siris syndrome Classification Unknown Unknown ‘Purely’ genetic ‘Purely’ Acquired Unknown ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ earing H impairment ------+ ------Developmental Developmental calender age at age 6 months at at 6 months 9+8 years Intellectual disability + + + + + + + + + + + + + + + + Gestational Gestational age 36 39 38 42 41 31 33 40 34 38 35 Birth weight (gram) 2325 2735 2500 3740 3450 4000 1525 1980 emale Male/ F Male Male Female Male Male Female Female Male Male Male Female Male Male Male Male Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information ID 109 122 120 119 110 128 124 126 127 118 115 113 111 112 117 116 A

54 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Chapter 2.1 NR I cerebrum abnormalities M R I cerebrum (based on reports) Enlarged supratentorial ventricle ventricle supratentorial Enlarged liquor and wide peripheral system intensity: of high signal Areas area. of periventricular normal or sign leukomalacia. loss of white Polymicrogyria, dilatation. with ventricle matter Hypomylelinisation Intraventriculair haemorrhage, haemorrhage, Intraventriculair leukomalacia periventricular Gliosis periventricular, white and white Gliosis periventricular, loss matter grey Periventricular leukomalacia Periventricular NR Periventricular leukomalacia Periventricular Normal Ventriculomegaly Normal NR isk R factor H PVL, S PVL PVL PVL Chromosomal Chromosomal on 10 aberration and 12 Genetic diagnosis Unknown Genetic Unknown Classification Unknown ‘Purely’ acquired ‘Purely’ Unknown Unknown ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown earing - + H impairment - - - - Developmental Developmental calender age at age IQ 67 2+6 years at at 2+6 years 3+8 years 8-9 months at at 8-9 months 2+3 years 21 months at at 21 months 40 months 16-33 months 16-33 months 3+6 years at + Intellectual disability + + + + + + + + + + + Gestational Gestational age 31 36 40 32 30 31 Birth weight (gram) 1503 2000 3470 2100 3950 1640 1105 emale Male Male/ F Male Female Female Male Male Female Male Male Female Male Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 135 134 136 133 ID 131 138 142 139 145 143 147 146 A

55 Chapter 2.1 Vigabatrin Yes Yes NR I cerebrum abnormalities M R I cerebrum (based on reports) Delayed myelination, wide myelination, Delayed ventricles Periventricular leukomalacia and Periventricular callosum thin corpus Dysgenesis, possibly lissencephaly Dysgenesis, Cysts periventricular Cysts NR NR NR NR Normal Chiari one malformation, Chiari one malformation, loss en white matter white Delayed abnormalities. matter myelination. Periventricles high signals in high signals Periventricles Hypoplastic corpus matter. white callosum Progressive atrophy of cerebellum atrophy Progressive isk R factor T PVL, S W PVL, W PVL, P PVL Genetic diagnosis Aromatic Aromatic decarboxylase deficiency Rett syndrome Trisomy 21 Trisomy Unknown ‘Purely’ acquired ‘Purely’ Classification ‘Purely’ acquired ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Acquired Genetic Genetic Unknown Genetic Unknown Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ earing - - H impairment ------+ - Developmental Developmental calender age at age 11 months at 2 at 11 months years 22 months at at 22 months 3+1 years 3-4 months at at 3-4 months 2+5 years 13-20 months 13-20 months 6+4 years at IQ <60 13 months at at 13 months 18 months Intellectual disability + + + + + + + + + + + + + Gestational Gestational age 37 38 33 40 37 32 40 39 34 2195 Birth weight (gram) 1650 1375 3500 3500 1660 2900 3100 2590 2120 emale Female Male/ F Male Male Female Male Female Female Female Female Male Female Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 157 158 156 152 ID 149 159 173 172 163 161 171 170 166 A

56 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Chapter 2.1 Normal NR I cerebrum abnormalities M R I cerebrum (based on reports) NR Normal NR Pachygyria and white matter matter and white Pachygyria abnormalities Holoprosencephaly NR NR Less myelination, corpus callosum corpus myelination, Less horns wide posterior agenesis, Bilateral hemorraghes, cystic hemorraghes, Bilateral leucoencephalopathy Cerebral necrosis, with subcortical necrosis, Cerebral cystic and leukomalacia, necrosis in thalamus and striatum. atrophy isk R factor H H, I S - D2-hydroxygluta araciduria Genetic diagnosis Trisomy 21 Trisomy Infantile neuroaxonal dystrophy Unknown Unknown Genetic Classification Unknown ‘Purely’ genetic ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Unknown Acquired ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown earing - - H impairment - + - - - - - IQ 48 Developmental Developmental calender age at age 1+3 years at 11 at 1+3 years years 7 months at at 7 months 3+4 years 10-12 months 10-12 months 5+10 years at 13 months at at 13 months 4+3 years 2 years at 3 at 2 years years + + Intellectual disability + + + + + + + + + + Gestational Gestational age 42 41 32 39 41 39 Birth weight (gram) 2900 2575 3025 3000 emale Male Male Male/ F Female Female Female Female Female Male Male Female Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 178 176 180 179 ID 174 175 181 187 185 184 189 188 A

57 Chapter 2.1 Vigabatrin Yes I cerebrum abnormalities M R I cerebrum (based on reports) Wide peripheral liquor areas and liquor areas peripheral Wide in the basal spaces Virchow-Robin atrophy ganglia, minimal cerebral Possible posterior infarction posterior left, Possible nucleus of lentiform hyperintensity Normal Hypertrophic left hemisphere, secondary atrophy NR NR Hyperintense leasions white occipital periventricular atrophy mild cerebellar matter, NR Corpu callosum agenesis, callosum agenesis, Corpu vermis cerebellar hypoplastic NR in clacarin’s mainly occipital Gliosis, frontal and fissure central till fissure vermis gliosis in cranial Also gyrus. cortex. Ulegyria and cerebellar Dysplastic callosum, wide corpus abnormal cerebellar ventricles, peduncles. isk R factor COM T Genetic diagnosis Pitt-Hopkins Pitt-Hopkins syndrome Classification Unknown ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown Unknown Unknown Unknown Unknown Unknown Unknown ‘Purely’ acquired ‘Purely’ Unknown earing H impairment - - - - - Developmental Developmental calender age at age 5 years at 6 at 5 years years 12 months at at 12 months 3+3 years Intellectual disability + + + + + + + + + + + + Gestational Gestational age 38 40 40 40 35 32 40 36 Birth weight (gram) 2980 3250 3555 3520 3200 1980 2895 2440 emale Male/ F Male Female Female Male Female Male Male Female Male Male Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 194 ID 192 199 200 202 203 205 207 208 210 211 212 A

58 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) Some atrophy and some delayed and some delayed Some atrophy myelination. Cytotoxic edema, small frontal edema, small frontal Cytotoxic bleeding Hypomyelination Normal NR Cortical abnormalities parieto- occipital atrophy cerebral Severe Vermis atrophy Vermis Large liquor areas, frontal and frontal liquor areas, Large temporal NR NR Normal Schizencephaly, calcifications, calcifications, Schizencephaly, cerebellum atrophy isk R factor W COM TT PVL Genetic diagnosis Pelizaeus- Merzbacher syndrome Opitz C syndrome Citrullinaemia Trisomy 21 Trisomy Trisomy 21 Trisomy Classification Hydrocephalus and/ Hydrocephalus syndrome West or Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ Genetic Unknown ‘Purely’ acquired ‘Purely’ Unknown Genetic ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ genetic ‘Purely’ ‘Purely’ genetic ‘Purely’ earing H impairment - + - - - - + Developmental Developmental calender age at age 1+3 years at 7 at 1+3 years years 2 years at 2+8 at 2 years years 2 months at 10 at 2 months months Intellectual disability + + + + + + + + + + + + + Gestational Gestational age 40 40 40 32 38 41 37 40 29 40 39 Birth weight (gram) 3240 3000 1900 2540 2310 3370 3500 3415 emale Male/ F Female Female Female Male Female Male Female Female Female Female Female Male Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information ID 213 214 220 219 215 216 224 225 226 221 223 229 228 A

59 Chapter 2.1 Vigabatrin Yes NR NR I cerebrum abnormalities M R I cerebrum (based on reports) Normal callosum, corpus Thin matter white periventricular Abnormal signal abnormalities. cerebral capsule untill internal from peduncles. Small cavum septum pellucidum. Small cavum NR Cysts, old ischaemia in both Cysts, spaces arachnoidal Enlarged ventricles and delayed and delayed ventricles Enlarged myelination Minor aspecific abnormalities occipital Abnormalities in basal ganglia. Occipital and parietalOccipital also abnormalities in abnormalities, basal ganglia and thalamus Normal isk R factor S PVL S W I, S Genetic diagnosis Marden-Walker Marden-Walker syndrome Trisomy 21 Trisomy Duplicaton Duplicaton Xq11.2-q22.3 Duplication Duplication 2q37, Deletion 2q37, Deletion 15q24.3-q26.3 Propion acidemia Propion Unknown Unknown ‘Purely’ acquired ‘Purely’ Classification ‘Purely’ acquired ‘Purely’ Genetic Unknown Genetic ‘Purely’ acquired ‘Purely’ Genetic ‘Purely’ genetic ‘Purely’ Genetic ‘Purely’ acquired ‘Purely’ earing - + H impairment - - - - + - - IQ 68-72 Developmental Developmental calender age at age 3+6 years at 8 at 3+6 years years 18 months at at 18 months 4+9 years IQ 77-89 30 months at at 30 months 3+6 months 3+7 years at at 3+7 years 5+7 years + Intellectual disability + + + + + + - + + + + Gestational Gestational age 38 28 35 40 34 39 37 36 3550 Birth weight (gram) 2115 2500 2040 1710 3325 2950 emale Male Female Male/ F Female Female Female Female Female Male Female Male Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 234 233 235 236 232 ID 231 244 243 242 241 238 245 A

60 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Yes Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) Hemimegacephaly Abnormal cerebellum, hypoplastic hypoplastic Abnormal cerebellum, abnormal lot of liquor, cerebellum, gyration. Wide peripheral liquor areas, liquor areas, peripheral Wide mainly frontotemporal. White matter loss along the matter White corpus Thin mainly left. ventricles, callosum. NR Partial hemispherectomy left side, left side, hemispherectomy Partial gliosis callosum agenesis, corpus diffuse area, along operation abnormalities in left matter white hemisphere. abnormalities around Cystic ventricles. NR NR corpus dysgenesis, Cerebral and callosum hypoplasia cerebri of the flax hypoplasia NR isk R factor H, W H, PVL PVL C, W C, W Genetic diagnosis Trisomy mosaic Trisomy 21 Unknown Classification Unknown Unknown Hydrocephalus and/ Hydrocephalus syndrome West or ‘Purely’ acquired ‘Purely’ genetic ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ Unknown Acquired and/ Hydrocephalus syndrome West or earing - H impairment + - - + - + Developmental Developmental calender age at age 9 months at 18 at 9 months months 8 months at 5 at 8 months years 2+9 years at at 2+9 years 3+7 years at 11 months 24 months 2-3 months at at 2-3 months 16 months Intellectual disability + + + + + + + + + + + Gestational Gestational age 40 39 38 39 41 31 33 41 Birth weight (gram) 3870 3970 3620 1685 2230 4750 emale Male/ F Male Male Female Male Male Male Male Female Female Male Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 249 ID 247 251 250 255 256 252 253 257 260 259 A

61 Chapter 2.1 Vigabatrin Yes Yes I cerebrum abnormalities M R I cerebrum (based on reports) Frontal pachygyria, thin corpus thin corpus pachygyria, Frontal callosum NR Wide sulci pattern, wide central wide central sulci pattern, Wide liquor areas, and peripheral myelination delayed callosum agenesis, Corpus heterotopia NR NR Periventricular leukomalacia with Periventricular cysts and occipital frontoparietal NR NR Corpus callosum hypoplasia Corpus NR Wide ventricle system, delayed delayed system, ventricle Wide left subependyma myelination, of diminished signal, small area of small possible remaining bleeding. isk R factor W W PVL, W PVL, PVL Genetic diagnosis Lissencephaly I and III Complex deficiency Aicardi syndrome Aicardi Duplication Duplication 9q34-qter, pter- Duplication 15q13 Classification Genetic Genetic Unknown Genetic Hydrocephalus and/ Hydrocephalus syndrome West or Acquired Unknown Unknown ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ genetic ‘Purely’ Unknown earing H impairment ------+ Developmental Developmental calender age at age 10-11 months 10-11 months 3+3 years at Verbal IQ 100 Verbal 1+6-2+6 years 1+6-2+6 years 5+7 years at 3+1 years at 5 at 3+1 years years 9 months at at 9 months 5+2 years Intellectual disability + + + - + + + + + + + + Gestational Gestational age 40 39 32 36 32 41 32 42 41 Birth weight (gram) 3100 3580 1845 2570 1800 3250 1800 3340 2700 emale Male/ F Female Female Male Female Female Male Female Male Male Male Male Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 266 267 ID 261 270 269 263 271 272 277 276 274 278 A

62 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) NR Severe tissue loss, mainly high tissue loss, Severe Multicysticparietal. leukomalacia. Subarachnoid haemorrhageSubarachnoid intracerebral with subsequent and of ischaemia mainly occipital of left Thrombosis the brainstem. sinuses sigmoid NR Periventricular leukomalacia with Periventricular cysts Intraventricular hemorraghe left hemorraghe Intraventricular and right side NR Increased liquor area frontal Increased liquor area Multicystic encephalopathy with Multicystic encephalopathy of basal ganglia necrosis Intracranial hemorraghe Intracranial Delayed myelination, dorsal thin myelination, Delayed callosum corpus Dandy Walker anomaly Walker Dandy isk R factor PVL COM I PVL S S PVL H, S Genetic diagnosis Deletion 3p Classification Genetic Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown Unknown acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Acquired earing H impairment - - - - - + - - - - Developmental Developmental calender age at age 7 months at 9 at 7 months years 7-8 months at at 7-8 months 16 months 2+3 years at at 2+3 years 4+4 years 5-6 months at 6 at 5-6 months years 6-12 months at at 6-12 months 2+3 years Intellectual disability + + + + + + + + + + + + Gestational Gestational age 37 29 42 40 27 27 40 39 38 Birth weight (gram) 2505 1120 3390 4070 1420 760 3600 3500 3000 emale Male/ F Female Male Female Male Female Male Male Male Male Male Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 287 286 282 ID 279 293 290 289 288 285 284 283 281 A

63 Chapter 2.1 Vigabatrin Yes Yes I cerebrum abnormalities M R I cerebrum (based on reports) NR NR Normal Periventricular leukomalacia Periventricular NR NR NR NR Ventral corpus callosum agenesia corpus Ventral ventricles. and enlarged NR Hydrocephalus, with cystic aspect, Hydrocephalus, callosum not visible corpus Hypomyelination, wide central wide central Hypomyelination, liquor areas. peripheral Wide ventricles, frontal pachygyria. frontal ventricles, Wide NR Normal NR callosum agenesis, Corpus right pachygyria heterotopia, temporal isk R factor S, T S, S PVL PVL W H W Genetic diagnosis Deletion 5p13 CDG type 1a Deletion 14q32, 7q36 Duplication 45,X/46,X,r(X) Aicardi syndrome Aicardi Classification ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown Unknown Unknown Unknown Genetic Unknown Genetic ‘Purely’ genetic ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Genetic earing H impairment - + - - - + ------Developmental Developmental calender age at age IQ 85 4-7+5 years at at 4-7+5 years 10+7 years 4-8 months at at 4-8 months 9+4 years 8 months at at 8 months 1+11 years 1+3 years at 9 at 1+3 years years 6-12 months at at 6-12 months 2+2years Intellectual disability - + + + + + + + + + + + + + + + + Gestational Gestational age 32 32 42 31 37 31 40 40 40 32 41 39 39 42 Birth weight (gram) 760 780 1650 2100 1970 3860 4750 2000 2650 2085 2700 3245 emale Male/ F Male Female Male Male Male Female Male Male Female Male Male Female Male Male Male Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 295 ID 294 301 299 297 296 303 304 306 314 313 312 311 307 310 305 316 A

64 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) Abnormal Wide ventricles and brain damage and brain ventricles Wide in both hemispheres. Ischaemic stroke and subdural and and subdural Ischaemic stroke hemorraghes parenchymal Atypical white matter matter white Atypical and in the abnormalities frontal area radiata corona NR Holoprosencephaly NR Wide lateral ventricles, diminished ventricles, lateral Wide matter white Delayed gyration with under gyration Delayed lobes of frontal development NR NR NR NR Old stroke in leftOld stroke insula and in thalamus calcifications isk R factor H, I S C S Genetic diagnosis ATR-X Copper storage storage Copper disorder Trisomy 21 Trisomy Duplicaton 17q Duplicaton Classification Unknown Acquired Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ genetic ‘Purely’ Unknown Unknown Unknown ‘Purely’ genetic ‘Purely’ ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ earing H impairment - - - - + - - - Developmental Developmental calender age at age Verbal IQ 66 Verbal 4-6 months at at 4-6 months 4+5 years <1 years at 7 at <1 years years 6-7 months at at 6-7 months 2+1 years 12-15 months 12-15 months 12 years at Intellectual disability + + + + + + + + + + + + + + Gestational Gestational age 38 31 41 41 42 38 37 39 40 39 39 Birth weight (gram) 3510 2280 4250 2425 3000 2010 3425 3250 4012 3000 emale Male/ F Female Male Female Female Male Male Female Male Male Male Female Female Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information ID 317 319 325 323 322 327 329 326 330 332 334 335 333 336 A

65 Chapter 2.1 Vigabatrin Yes Yes I cerebrum abnormalities M R I cerebrum (based on reports) Long velum medullary velum Long posterior NR NR NR NR due to Irregular wide ventricles in white Calcifications tissue loss. also in grey and occipital matter cysts Arachnoidal in the matter. left cerebellar fossa posterior Cerebral hemispheral white matter matter white hemispheral Cerebral Cerebellar almost disappeared. atrophy High in thalamus and globus signal centre and in semioval pallidus, A bit sulcus. and along the central in in optic radiations higher signal matter. white the occipital NR NR isk R factor H P COM P S Genetic diagnosis Trisomy 21 Trisomy Tubereus sclerosis Vanishing white white Vanishing matter Classification Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Genetic and acquired Unknown Genetic and acquired Unknown Unknown Genetic ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ earing H impairment + - Developmental Developmental calender age at age 3 months at 18 at 3 months months 8-9 months at at 8-9 months 18 months at 10 months 13+4 years at 15 months 38 months 6-7 months at at 6-7 months 3+4 years Intellectual disability + + + + + + + + + + Gestational Gestational age 39 42 40 40 39 41 40 42 Birth weight (gram) 2285 4200 2980 3450 3650 4250 emale Male/ F Female Female Male Male Female Male Male Male Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information ID 337 338 339 340 342 341 344 349 347 348 A

66 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) NR Normal Corpus callosum agenesis. Delayed Delayed callosum agenesis. Corpus possible frontal, myelination, dysgyria parietal and cranial Wide ventricles, partial ventricles, corpus Wide callosum agenesia NR Parenchymal hemoraghe left hemoraghe Parenchymal edema left hemisphere temporal, with possible ischaemia. Cortical atrophy NR NR Wide ventricles, periventricular periventricular ventricles, Wide left abnormalities, matter white than right side. severe more Polymicrogyria, abnormal Polymicrogyria, diminished myelination, periventricular, matter white matter, in white calcifications dysgyria. cerebellar isk R factor S H, S PVL, S S, W S, C Genetic diagnosis Duplication 13q Duplication Rett syndrome Classification Unknown ‘Purely’ genetic ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ Unknown Unknown Acquired ‘Purely’ acquired ‘Purely’ Genetic Acquired ‘Purely’ acquired ‘Purely’ earing H impairment - + - - - - Developmental Developmental calender age at age 3-6 months at at 3-6 months 10 months 1 year at 10 at 1 year years Intellectual disability + + + - + + + + + + + Gestational Gestational age 41 39 28 40 39 34 31 30 Birth weight (gram) 3800 3080 1548 3470 3800 3060 1260 emale Male/ F Female Male Male Male Male Male Male Male Male Female Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 353 ID 352 359 356 360 361 362 366 365 364 363 A

67 Chapter 2.1 Vigabatrin Yes I cerebrum abnormalities M R I cerebrum (based on reports) Prominent posterior horns posterior of the Prominent diminshed occipital ventricles, matter white Abnormal myelination pattern pattern Abnormal myelination age. for Diminished gyri, asymmetric system. ventricle Wide cisterna magna. Wide Wide magna. cisterna Wide system. ventricle Wide ventricles Wide Wide ventricles with drain in situ, in situ, with drain ventricles Wide pachygyria. Bad quality, not possible to asses not possible to Bad quality, Normal structures. matter. white NR Normal Severe periventricular periventricular Severe matter, abnormalities of the white cysts Wide liquor areas, arachnoidal cyst arachnoidal liquor areas, Wide matter white Possible left temporal. mainly left. centre, loss in semioval Small frontal lobes Small frontal isk R factor S H PVL Genetic diagnosis Complex I Complex deficiency Classification ‘Purely’ acquired ‘Purely’ Unknown Unknown Unknown Unknown Unknown Unknown Unknown Hydrocephalus and/ Hydrocephalus syndrome West or ‘Purely’ genetic ‘Purely’ ‘Purely’ acquired ‘Purely’ Unknown earing H impairment ------Developmental Developmental calender age at age 9-11 months at at 9-11 months 7+9 years 12-18 months 12-18 months 11 years at 9-10 months at at 9-10 months 3+5 years 2-2+6 years at at 2-2+6 years 17 years at 2-2+6 years 7+9 months 12-18 months 12-18 months 4 years at 3 months at 6 at 3 months years 13-16 months 13-16 months 22 months at 2+8 years at at 2+8 years 3+8 years Intellectual disability + + + + + + + + + + + + Gestational Gestational age 39 27 40 37 39 41 36 41 40 40 Birth weight (gram) 3030 1150 2750 2560 3170 2707 2525 3100 4410 3810 emale Male/ F Male Male Male Male Female Female Male Female Female Male Male Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 368 ID 367 370 371 373 376 386 382 383 384 378 377 A

68 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Yes Yes Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) NR Normal Severe multicystic encephalopathy. multicystic encephalopathy. Severe and central peripheral Wide callosum. corpus Thin liquorareas. Ischaemia of almost whole left white hemisphere cerebral leftSome calcifications occipital. left basal ganglia and matter at cortical left frontobasal Corpus callosum agenesis, possible callosum agenesis, Corpus partial falx agenesis. Frontal and left occipital white and left white occipital Frontal Wide abnormalities. matter and some ventricle ventricles asymmetry Hydrocephalus and delayed and delayed Hydrocephalus myelination Wide ventricles and some ventricles Wide of subarachnoidal enlargement Atrophy? spaces. Cortical thin corpus dysplasia, of myelination callosum, delayed right hemisphere. Normal isk R factor PVL S S W C H Genetic diagnosis Deletion 17q21.31 Complex II Complex deficiency Classification ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or ‘Purely’ acquired ‘Purely’ Unknown Unknown Genetic ‘Purely’ genetic ‘Purely’ earing H impairment - - - - Developmental Developmental calender age at age 14 months at at 14 months 3+3 years 9 months at 4 at 9 months years 2+6 years at at 2+6 years 3+4 years 6 months at at 6 months 2+10 years 4 months at 7 at 4 months months Intellectual disability + + + + + + + + + + Gestational Gestational age 38 32 38 40 36 42 42 41 41 Birth weight (gram) 2210 1740 1860 2700 2500 3715 3360 2900 3650 emale Male/ F Male Male Male Male Male Male Female Male Male Male dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 388 ID 387 389 398 397 395 391 401 403 402 A

69 Chapter 2.1 Vigabatrin Yes Yes I cerebrum abnormalities M R I cerebrum (based on reports) NR Abnormal white matter matter Abnormal white and mainly temporal signal, in semi-ovalperiventricular centre. than left affected Right side more cysts matter. Several in white side. thin aspect of cerebellum, Atrophy of cortex, dimished gyrations. NR NR Necrosis of basal ganglia, brain of basal ganglia, brain Necrosis stam abnormalities NR White matter loss matter White NR Normal Right temporal parietal occipital parietal occipital Right temporal stroke NR NR Possible atrophy cerebellum atrophy Possible isk R factor S C H H, I P W S H, I Genetic diagnosis Classification Unknown ‘Purely’ acquired ‘Purely’ Unknown ‘Purely’ acquired ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Acquired ‘Purely’ acquired ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Unknown ‘Purely’ acquired ‘Purely’ Acquired earing H impairment - - + + - + + - Developmental Developmental calender age at age 12-15 months 12-15 months 17 years at IQ 55 9 months at at 9 months 1+7 years Intellectual disability + + + + + + + + + + + + + Gestational Gestational age 34 31 30 39 34 40 42 39 Birth weight (gram) 1470 880 2105 2200 4200 3146 3380 2740 emale Male/ F Female Male Male Female Male Male Female Male Male Male Male Male Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information ID 407 420 417 416 413 408 422 421 414 412 410 411 409 A

70 Low vision due to CVI: differentiating between acquired and genetic causes Vigabatrin Yes Yes Yes Yes Yes Chapter 2.1 I cerebrum abnormalities M R I cerebrum (based on reports) Normal NR hornAbnormal posterior right. typeLissencephaly 1 Severe abnormalities in basal Severe and central ganglia, thalamus area gyrus. NR NR State after resection of occipital State hydrocephalus encephalocele, Normal Delayed myelination Delayed Ischaemic abnormalities diffuse periventricular Severe cysts, leukomalacia. Normal Holoprosencephaly isk R factor W P I H W COM, S, W S, COM, W PVL, Genetic diagnosis Deletion 22q13, Duplication 15q22-qter Complex I Complex deficiency Incontinentia Incontinentia Pigmenti Unknown Unknown Classification Hydrocephalus and/ Hydrocephalus syndrome West or Genetic ‘Purely’ acquired ‘Purely’ Unknown Genetic ‘Purely’ acquired ‘Purely’ Hydrocephalus and/ Hydrocephalus syndrome West or Unknown Unknown Hydrocephalus and/ Hydrocephalus syndrome West or Genetic and acquired Acquired earing - - H impairment - - - - Developmental Developmental calender age at age 5-6 months at at 5-6 months 5+6 years 5-6 months at at 5-6 months 3+4 years 15-18 months 15-18 months 1+9 years at Intellectual disability + + + + + + + + + + + + + - Gestational Gestational age 42 36 32 39 29 40 Birth weight (gram) 3350 2230 2200 3170 1557 3945 emale Male/ F Female Male Male Male Male Female Female Male Male Male Male Female Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 445 443 440 441 424 ID 423 438 433 432 447 429 428 426 425 A

71 Chapter 2.1 Vigabatrin Yes I cerebrum abnormalities M R I cerebrum (based on reports) Large ventricles. Liquorflow ventricles. Large stoma. through NR Enlarged lateral ventricles. Atrophy. Atrophy. ventricles. lateral Enlarged ischaemia. Delayed frontal Left myelination. NR NR Necrosis of basal ganglia, thalamus Necrosis atrophy. Cerebral stem. and brain NR Normal isk R factor H, I S S C P, W P, COM Genetic diagnosis Unknown Classification Unknown Acquired ‘Purely’ acquired ‘Purely’ acquired ‘Purely’ ‘Purely’ acquired ‘Purely’ Acquired ‘Purely’ acquired ‘Purely’ earing - H impairment - - - Developmental Developmental calender age at age 15-18 months 15-18 months 4+4 years at IQ 57 < 1 year at 3+4 at < 1 year years IQ 91 Intellectual disability - + + + + + + - Gestational Gestational age 42 37 36 27 36 40 Birth weight (gram) 2900 3490 1200 770 3950 3000 3860 emale Male/ F Male Male Female Male Male Female Female Female dditional file 1: Phenotype information of all patients involved, part 1 (continued) involved, of all patients dditional file 1: Phenotype information 454 452 450 451 ID 448 458 457 456 A S=stroke, leukomalacia, PVL=periventricular problems, P=perinatal reported, not NR= I=menigitis/encephalitis, H=hydrocephalus, COM=complication, infection, C=CMV TT=twin-twin T=trauma, syndrome tranfusion

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes

dominates

uditive stimuli stimuli uditive A Yes Yes Yes Yes Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes

taring at light at taring S

Yes Yes Yes Yes Crowding

Yes Pale optic discs optic Pale

No No No No No Yes No No Yes No No Yes No No No No Yes

ystagmus N

Yes No Yes Yes Yes No No No No Yes Yes No No No Yes Yes Yes

disorder

ye movement movement ye E

No No Yes No No No No No Yes No Yes No No No No Yes

trabismus S

No Yes Yes Yes Yes No Yes No Yes Yes Yes Yes Yes No Yes Yes Yes Visual field defects field Visual

Yes Yes Yes Yes No No Yes No Yes No Yes Yes Yes

S D O acuity Visual

LP 0.02 0.2 0.25 0.15 0.2 0.07 0.3 LP 0.3 0.3 0.25 0.2 0.25 0.2 0.25 0.04 LP

OS error efraction R

S+5.00 S+3.00 till S+1.00 S+1.00=C-4.75x178’ S+3.00=C-1.00x0” S+2.00 S+0.50=C-0.50x180” S+2.50=C-1.00x180” S+1.25 S-2.50=C-3.50x2”

D O

efraction error error efraction R

S+5.00 S+3.00 till S+1.00 S+1.75=C-1.75x20” S+1.75=C- 1.00x180” S+2.00 S+1.00 S+3.00=C- 1.50x130” S+1.25 S+1.00=C-4.50x12”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

17 m 12 m 42 m 44 m 13 m 34 m 9 m 6 y 26 m 34 m 24 m 8 y 27 m 6 y 4 y 6 y 20 m 7 m ID dditional file 1: Phenotype information of all patients involved, part 2 involved, of all patients dditional file 1: Phenotype information 25 22 20 19 18 17 16 15 12 11 9 7 6 5 4 3 2 1 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes

dominates

uditive stimuli stimuli uditive A

Yes Yes

the target the

ooking away from from away ooking L

Yes Yes Yes Yes Yes Yes Yes Yes Yes

taring at light at taring S

Yes Yes Yes Yes Yes Yes

Crowding Pale optic discs optic Pale

No Yes No Yes No No No No No No Yes No No No No No Yes

ystagmus N

Yes Yes Yes Yes Yes No No No No Yes No No No No No Yes No No Yes

disorder

ye movement movement ye E

Yes Yes No Yes No No No Yes No No No No No No Yes Yes No No Yes

trabismus S

Yes Yes Yes Yes Yes No No No No No No Yes Yes No No No Yes Yes Yes No Visual field defects field Visual

Yes Yes Yes No No Yes No Yes No Yes No Yes No Yes Yes No

S D O acuity Visual

0.01 0.05 0.08 0.03 0.2 0.25 0.3 0.04 0.25 0.12 0.15 0.2 0.25 0.25 0.3 0.07 0.2 0.15 0.03 0.1

OS error efraction R

S+1.00=C-0.50x180” S+1.75=C-2.00x180” S+1.75 C-3.00x180” S+2.00=C-1.00x90” S+8.00 S+0.50 S+5.00

D O

efraction error error efraction R

S+2.00=C-2.00x20” S+1.75=C-2.5x180” S+1.75 C-3.00x180” S+1.00=C-1.00x90” S+8.00 S+0.50 S+5.00

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

32 m 27 m 7 y 10 m 47 m 10 y 4 y 5 m 13 y 44 m 27 m 24 m 4 y 44 m 5 y 12 m 4 y 4 y 27 m 27 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 50 48 49 47 46 45 43 42 41 40 39 37 34 36 33 32 31 29 28 27 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes

dominates

uditive stimuli stimuli uditive A Yes Yes Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes

taring at light at taring S

Yes Yes Crowding

Yes Yes Yes Pale optic discs optic Pale

No No No No No No Yes No No Yes Yes Yes Yes No No Yes

ystagmus N

No No No Yes Yes No Yes No Yes Yes No Yes Yes Yes No No

disorder

ye movement movement ye E

No Yes No No No No Yes Yes No No No No No No No No

trabismus S

Yes No Yes Yes Yes No Yes Yes Yes Yes No Yes Yes Yes Yes Yes Visual field defects field Visual

Yes No Yes No No No No Yes No No Yes Yes No No

S D O acuity Visual

0.3 0.01 0.1 0.02 0.25 0.3 0.16 0.02 0.15 0.04 0.3 0.125 LP 0.15 0.05 0.04 0.25 LP

OS error efraction R

S+6.00=C-1.25x70” S+1.50=C-0.50x180” S+2.50=C-5.00x10” S-1.00=C-4.00x16” S+2.00 S+8.00 S+3.00 S+1.00=C-1.00x90” S-1.00=C-4.00x180” S-3.25=C-1.00x45” S+3.00

D O

efraction error error efraction R

S+6.25=C- 1.25x105” S+1.00 S+2.25=C- 0.25x168” S-1.75=C-2.00x160” S+2.00 S+8.00 S+3.00 S+1.00=C-1.00x90” S+3.00=C-3.25x10” S-1.75=C-0.25x0” S+3.00

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

4 y 33 m 23 m 23 m 13 y 5 y 8 y 8 m 29 m 4 y 41 m 41 m 15 m 4 y 15 m 22 m 4 y 15 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 81 79 78 77 75 73 74 72 70 68 67 66 64 57 56 55 54 51 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes

dominates

uditive stimuli stimuli uditive A

Yes Yes Yes

the target the

ooking away from from away ooking L

Yes Yes Yes Yes

taring at light at taring S

Yes Yes Yes Yes Yes Crowding

Yes Yes Pale optic discs optic Pale

Yes No No Yes Yes No Yes No Yes No Yes Yes No Yes No Yes

ystagmus N

No Yes Yes Yes Yes Yes Yes No No No No No Yes No Yes No

disorder

ye movement movement ye E

No No Yes No No Yes Yes No Yes Yes Yes No No Yes Yes Yes

trabismus S

Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Visual field defects field Visual

Yes No Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

S D O acuity Visual

0.25 0.2 0.2 0.08 0.1 No LP 0.1 0.07 0.3 0.03 0.02 0.15 LP 0.3 0.3 0.07 0.03 0.3

OS error efraction R

S+9.00 S-2.00 S+1.00=C-6.00x180” S+0.50 S-2.00 S+1.75=C-0.50x180” S+0.25=C-1.50x176” S+2.50=C-2.75x170”

D O

efraction error error efraction R

S+8.50 S-5.00 S+2.00=C-6.60x20” S+0.50 S-2.00 S+1.50=C- 0.75x180” S+0.50=C-1.75x9” S+2.25=C-2.50x10”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

25 m 4 y 30 m 21 m 8 y 34 m 20 m 8 y 20 m 7 y 18 m 19 m 14 m 44 m 10 y 30 m 23 m 5 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 107 105 104 103 102 101 99 97 96 95 93 92 88 89 87 85 84 83 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes Yes

dominates

uditive stimuli stimuli uditive A Yes Yes Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes Yes Yes

taring at light at taring S

Yes Yes Yes Crowding

Yes Yes Yes Pale optic discs optic Pale

Yes Yes Yes No Yes No No No No Yes No Yes No No No Yes

ystagmus N

No Yes Yes No No Yes No No No No No No No

disorder

ye movement movement ye E

No No Yes No No No No Yes No No Yes No Yes Yes Yes No

trabismus S

No No Yes Yes Yes No Yes Yes Yes Yes No Yes Yes Yes No Visual field defects field Visual

No No Yes Yes No No Yes Yes Yes Yes Yes No Yes Yes No Yes No

S D O acuity Visual

0.3 0.25 0.07 0.2 0.06 0.03 0.15 0.15 0.3 0.1 0.15 0.3 0.23 0.1 0.15 0.04 0.2

OS error efraction R

S+3.00 S+1.00=C-2.00x180” S+1.50=C-4.50x180” S+6.00=C-2.00x180” S+2.00=C-2.75x174” S+5.25=C-0.75x90” S+5.50=C-2.00x150” S-1.50=C-1.75x160”

D O

efraction error error efraction R

S+3.00 S+1.50=C- 2.50x180” S+1.50=C- 4.50x180” S+6.00=C- 2.00x180” S+4.00=C-4.00x5” S+5.50=C-0.75x90” S+5.00=C-2.00x85” S+0.25=C- 2.00x172”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

7 y 46 m 7 y 36 m 4 y 8 m 17 m 41 m 44 m 28 m 8 y 6 y 40 m 13 y 6 y 29 m 5 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 128 127 124 126 122 120 119 118 117 116 112 113 115 110 111 109 108 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes

dominates

uditive stimuli stimuli uditive A

Yes

the target the

ooking away from from away ooking L

Yes Yes Yes Yes

taring at light at taring S

Yes Yes Crowding

Yes Pale optic discs optic Pale

No Yes No No No Yes Yes No No Yes No Yes Yes No No No Yes Yes

ystagmus N

No Yes No Yes Yes Yes Yes No Yes Yes Yes No No No

disorder

ye movement movement ye E

No No No No No No Yes Yes No No No Yes Yes No No No

trabismus S

No Yes Yes Yes Yes No Yes Yes No Yes No Yes Yes No Yes No No Yes Visual field defects field Visual

Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes No No Yes

S D O acuity Visual

0.2 0.1 0.25 0.2 0.25 0.07 0.26 0.3 0.01 0.15 0.3 0.25 0.25 0.15 0.0125 0.07 0.2 0.25

OS error efraction R

S-0.75=C-2.50x175” C-2.00x180” S-2.00=C-0.50x180” S+2.50 S+1.00=C-2.00x180” S+3.00 S+1.75=C-2.50x20” S+1.00=C-1.25x147” S+2.50=C-1.50x90” S+2.50=C-1.50x5”

D O

efraction error error efraction R

S-0.50=C-2.25x180” C-2.00x180” S-1.50=C-0.50x180” S+3.00=C- 0.50x180” S+1.00=C- 3.00x180” S+4.00 S+2.25=C-1.50x15” S+1.75=C-2.50x20” S+2.50=C-1.50x90” S+3.00=C- 2.00x170”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

37 m 13 m 5 y 5 y 39 m 25 m 9 y 40 m 14 m 6 y 4 y 6 y 7 y 4 y 10 m 9 y 5 y 40 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 159 158 157 156 152 149 147 146 145 143 142 139 138 136 135 134 133 131 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes Yes

dominates

uditive stimuli stimuli uditive A Yes Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes

taring at light at taring S

Yes Yes Yes Yes Crowding

Yes Yes Pale optic discs optic Pale

Yes No No Yes Yes No No No No No Yes No No No Yes Yes No No

ystagmus N

Yes Yes No Yes No No Yes Yes Yes No Yes No Yes No No Yes

disorder

ye movement movement ye E

No No Yes Yes No No No No No Yes No No Yes No Yes

trabismus S

Yes Yes No Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Visual field defects field Visual

No No No Yes Yes Yes No Yes Yes Yes Yes No No Yes No Yes

S D O acuity Visual

0.12 LP 0.15 0.02 0.03 0.3 0.15 0.1 0.2 0.05 0.25 0.1 0.01 0.1 0.16 0.3 0.25 0.2

OS error efraction R

S+2.00 S-2.00=C-4.00x125” S+5.50=C-0.50x180” S+6.00=C-1.25x53” S+1.50=C-0.50x180” S+3.00=C-1.00x180” S+3.00 S-5.00 S+7.00=C-3.00x170”

D O

efraction error error efraction R

S+1.50 C-4.00x45” S+4.75=C- 0.5.00x180” S+5.75=C- 5.00x152” S-2.00 S+3.00=C- 1.00x180” S+3.00 S-6.00 S+5.00=C-1.25x5”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

4 y 9 y 7 y 45 m 13 m 8 y 5 y 7 y 5 y 20 m 9 y 38 m 14 m 18 y 6 y 36 m 6 y 13 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 188 187 185 184 181 180 179 176 178 175 174 173 172 171 170 166 163 161 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes Yes

dominates

uditive stimuli stimuli uditive A

Yes

the target the

ooking away from from away ooking L

taring at light at taring S

Yes Yes Yes Yes

Crowding Pale optic discs optic Pale

No Yes No Yes No Yes No No No No Yes No Yes No No No No No Yes

ystagmus N

No Yes No No No Yes No No Yes No Yes No No Yes

disorder

ye movement movement ye E

No No No No No Yes No No No No Yes Yes Yes Yes Yes No

trabismus S

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Visual field defects field Visual

Yes Yes No No No No Yes Yes Yes No Yes No Yes Yes

S D O acuity Visual

0.04 0.04 0.2 0.1 0.15 0.13 0.15 0.01 0.3 0.15 0.08 0.15 0.01 0.2 0.01 0.1 0.3 0.25 0.2

OS error efraction R

S+3.00 S-0.50=C-1.50x180” S+4.00 Myopia S+0.25=C-1.25x5” S+2.25=C-2.00x0” S+4.50=C-1.50x180” S+2.50=C-0.50x180”

D O

efraction error error efraction R

S+3.00 S-0.50=C-1.75x180” S+4.00 Myopia S+0.75=C-2.00x20” S-0.25=C-3.75x20” S+4.50=C- 1.00x180” S+2.00=C-0.50x10”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

43 m 25 m 27 m 16 y 15 m 23 m 4 y 21 y 6 y 35 m 11 y 7 y 4 y 47 m 16 m 22 y 11 y 35 m 14 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 220 219 216 215 214 213 212 210 211 208 207 205 202 203 200 199 194 192 189 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes Yes Yes Yes Yes

taring at light at taring S

Yes Crowding

Yes Yes Yes Pale optic discs optic Pale

No No Yes No No No Yes Yes No No Yes No Yes Yes Yes

ystagmus N

Yes No No No Yes No Yes Yes No No Yes Yes No No

disorder

ye movement movement ye E

Yes No No Yes Yes No Yes No No Yes Yes No No No

trabismus S

Yes No Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Visual field defects field Visual

No Yes No Yes No Yes No No No No Yes Yes

S D O acuity Visual

Possible Possible LP 0.3 0.13 0.3 0.05 0.12 0.2 0.3 0.15 0.15 0.3 LP LP 0.01 0.02 0.1 0.3

OS error efraction R

S+6.00=C-1.25x30” S+6.00=C-3.25x155” S-2.00=C-4.00x180” S-7.00 Hypermetropia S+3.00 S+6.25 S+4.50 S+2.50=C-1.00x180” S+8.00=C-2.00x180” S+5.75 S+4.50=C-1.00x21” S+3.50=C-0.50x6”

D O

efraction error error efraction R

S+6.00=C- 2.00x157” S+6.25=C-3.00x6” S-2.00=C-4.00x180” S-10.00 S+1.00 S+3.00 S+6.00=C- 0.50x175” S+6.00=C-1.50x90” S+2.50=C- 1.50x180” S+5.00 S+5.00 S+4.50=C-1.00x4” S+2.75

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

22 m 5 y 47 m 9 y 21 m 17 y 6 y 6 y 21 m 6 y 7 y 7 m 15 m 4 y 28 m 6 y 6 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 242 243 241 238 236 235 233 234 232 231 229 228 226 225 224 223 221 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A

Yes Yes Yes

the target the

ooking away from from away ooking L

Yes

taring at light at taring S

Yes Yes Yes Crowding

Yes Yes Pale optic discs optic Pale

No No No Yes Yes Yes No No No No Yes Yes No No No No

ystagmus N

No No Yes No Yes No Yes No No No Yes Yes Yes No No

disorder

ye movement movement ye E

Yes No No Yes No No Yes No No No No Yes Yes Yes No No

trabismus S

Yes No Yes Yes No No Yes Yes No No Yes Yes Yes Yes No No Visual field defects field Visual

No No No Yes No No Yes Yes Yes Yes Yes No Yes

S D O acuity Visual

0.2 0.15 0.03 0.2 0.2 0.15 0.02 0.1 0.25 0.3 0.1 0.15 0.25 0.007 0.2 0.3 0.3 0.1

OS error efraction R

S+3.00=C1.50x180” S+2.50=C-1.50x90” C-2.00x180” S+5.50=C-0.75x177” S+1.75 S+2.00 S+3.00 S+1.75 S+3.50=C-3.50x160” S+2.25=C-0.75x30”

D O

efraction error error efraction R

S+2.50=C- 1.00x180” S+3.00=C-1.50x45” C-2.00x180” S+5.50=C- 1.00x177” S+1.75 S+2.00 S+3.00 S+1.75 S+3.75=C-3.75x25” S+3.00=C-1.75x40”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

4 y 22 m 4 y 38 m 6 y 6 y 14 m 5 y 42 m 7 y 21 m 5 y 5 y 20 m 46 m 35 m 6 y 28 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 269 267 266 263 261 260 259 257 256 255 253 251 252 250 247 249 245 244 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes

dominates

uditive stimuli stimuli uditive A Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes

taring at light at taring S

Yes Crowding

Yes Pale optic discs optic Pale

Yes Yes No Yes No Yes No No Yes Yes Yes No Yes No Yes Yes

ystagmus N

Yes Yes No No Yes Yes Yes Yes No No No Yes No Yes No

disorder

ye movement movement ye E

No Yes No No Yes Yes Yes Yes No No No Yes No No No No No

trabismus S

No Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes No Yes Yes Visual field defects field Visual

Yes Yes No No Yes Yes Yes Yes Yes Yes No No Yes

S D O acuity Visual

0.05 0.2 0.1 0.3 0.16 LP 0.15 0.3 0.01 0.1 0.2 0.25 0.3 0.2 0.15 0.1 0.07

OS error efraction R

S+3.50=C-1.50x85” C+0.50x180” S-3.00 S+2,00=C-2.00x180” S+3.00 A bit hypermetropia S-3.25=C-1.25x44” S+0.75=C-1.00x180” S-0.75 S+2.00=C-1.00x180”

D O

efraction error error efraction R

S+2.50=C-0.50x80” S-3.00 S+1.00=C- 2.00x180” S+5.00 A bit hypermetropia S-3.50=C-0.50x75’” S+1.00=C1.25x180” S-0.50=C-0.50x180” S+2.00=C- 1.00x180”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

7 y 26 m 40 m 6 y 20 y 35 m 8 y 18 y 33 m 7 y 6 y 10 y 10 y 5 y 5 m 6 y 43 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 289 288 287 286 285 284 282 283 281 279 278 277 276 274 272 271 270 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes

dominates

uditive stimuli stimuli uditive A

the target the

ooking away from from away ooking L

taring at light at taring S

Yes Crowding

Yes Yes Pale optic discs optic Pale

Yes No No Yes No Yes Yes Yes No No Yes Yes No Yes Yes Yes No Yes

ystagmus N

No No No Yes No No No No No Yes No No Yes No No Yes

disorder

ye movement movement ye E

No Yes No No Yes No No No Yes Yes No Yes Yes No Yes Yes

trabismus S

Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes No No Yes Visual field defects field Visual

Yes Yes No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes

S D O acuity Visual

0.15 0.3 0.2 LP 0.25 0.15 0.03 0.05 0.3 0.08 0.1 0.015 0.15 0.25 0.3 0.3 0.05 0.25

OS error efraction R

S-9.00 S+1.50=C-3.50x180” S+0.25=C-0.50x1” S-7.00 S+2.00=C-2.00x180” S+5.00=C-1.00x180” S-1.00 S-2.25=C-1.50x30” S+1.75 S+3.00

D O

efraction error error efraction R

S-10.00 S+2.00=C- 4.00x135” S+0.25=C- 0.75x179” S-7.00 S+1.00=C- 1.00x180” S+4.00 S+1.00 S-1.75=C-0.75x160” S+1.75 S+3.00

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

14 y 5 y 11 y 8 y 39 m 45 y 33 m 25 m 4 y 9 y 8 y 15 m 23 m 30 m 10 y 15 y 12 y 4 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 314 313 312 311 310 307 306 305 304 303 301 299 297 296 295 294 293 290 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes Yes Yes

taring at light at taring S

Yes

Crowding Pale optic discs optic Pale

No No No Yes No No No Yes Yes Yes Yes Yes No No No

ystagmus N

No No Yes No No Yes No Yes No No Yes Yes Yes Yes No

disorder

ye movement movement ye E

No No No No No Yes No Yes No Yes Yes Yes Yes No

trabismus S

Yes No No Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes Visual field defects field Visual

Yes No Yes No No Yes Yes No Yes No Yes Yes No

S D O acuity Visual

0.2 0.13 0.3 0.2 0.1 0.15 0.15 0.01 0.25 No LP 0.01 0.3 0.08 0.2 0.3

OS error efraction R

S+2.00=C-1.00x45” S+2.00=C-1.50x25” S+3.00 S+3.50=C-0.50x180” S+1.00 S+3.00=C-4.00x180” S-7.00=C-3.00x180” S-8.50=C-7.50x180” S+1.50=C-2.50x180” S-2.00 S+4.25=C-1.50x15”

D O

efraction error error efraction R

S+2.00 S+2.50=C- 0.50x150” S+3.00=C- 0.50x180” S+3.00=C- 1.00x180” S-1.00 S+2.00=C- 3.00x180” S-7.00 S-7.50=C-4.00x180” S+2.00=C- 1.50x180” S-3.00 S+4.75=C- 1.75x179”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

18 m 6 y 29 y 12 y 7 y 5 y 44 m 6 y 17 y 27 m 30 m 5 y 9 y 22 y 9 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 336 335 334 333 332 330 329 327 326 325 323 319 322 317 316 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A

Yes Yes Yes

the target the

ooking away from from away ooking L

Yes Yes Yes Yes

taring at light at taring S

Yes Yes

Crowding Pale optic discs optic Pale

Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes No No No Yes

ystagmus N

No No No Yes No Yes No Yes No No Yes No No Yes Yes

disorder

ye movement movement ye E

Yes No No No No Yes No No No No No No No No No No Yes No

trabismus S

No No Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes No Yes No Yes Yes Visual field defects field Visual

Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes No

S D O acuity Visual

0.2 0.1 0.08 LP 0.06 0.1 0.05 0.03 LP 0.3 0.07 0.2 0.15 0.15 0.08 0.2 0.2 0.1 0.05

OS error efraction R

S+2.50 S-2.00 S+4.00=C-1.00x90” S-3.00 S+2.00 S+1.00=C-4.00x180” S+2.00 C-4.50x170” S-0.75=C-2.75x160” S-5.00 S+1.50

D O

efraction error error efraction R

S+2.50 S-2.00 S+3.00 S-3.00 S+2.00 S+2.00=C- 5.00x180” S+2.00 C-4.50x28” C-2.25x40” S-10.00 S+1.50

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

16 y 18 m 5 y 4 y 24 m 33 m 7 y 22 m 9 m 6 y 28 m 7 y 38 m 5 y 14 y 41 m 41 m 10 y 6 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 364 363 362 361 360 359 356 353 352 349 348 347 344 342 341 340 338 339 337 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/ Problems with with Problems

Yes Yes

dominates

uditive stimuli stimuli uditive A

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes Yes

taring at light at taring S

Yes Yes Yes Yes Crowding

Yes Yes Pale optic discs optic Pale

Yes No Yes Yes No Yes Yes No Yes No No Yes No No Yes Yes Yes No

ystagmus N

Yes No No Yes Yes Yes No No No No No Yes Yes No Yes Yes No

disorder

ye movement movement ye E

No Yes No Yes Yes No No Yes No No No No No Yes No Yes No No

trabismus S

No No Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No Visual field defects field Visual

No No Yes Yes Yes Yes Yes Yes No No No Yes Yes Yes No

S D O acuity Visual

0.01 0.02 0.15 0.13 0.15 0.2 0.2 0.05 0.25 0.16 0.2 0.3 0.2 0.15 0.15 0.1 0.1 0.07

OS error efraction R

S+3.00 S+1.00 S+2.00 S+5.00=C-3.00x7” S-0.50=C-2.50x45” S+8.00=C-1.00x180” S+2.50=C-2.00x180” S-4.00=C-5.00x180” S+2.75 S+2.50 S+1.00 S+2.00

D O

efraction error error efraction R

S+2.50=C-1.00x0” S+2.00 S+2.00 S+7.00=C-4.50x10” S-1.00=C-3.00x135” S+8.00=C- 1.00x180” S+2.75=C- 1.75x180” S-9.00=C-3.00x180” S+3.25 S+2.00 S+1.00 S+2.00

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

26 m 17 m 5 y 6 y 8 y 14 y 14 y 17 y 35 m 9 y 36 m 9 y 17 y 7 y 4 y 7 y 8 y 9 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 391 389 388 387 386 384 383 382 378 377 376 373 371 370 368 367 366 365 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A

the target the

ooking away from from away ooking L

Yes Yes

taring at light at taring S

Yes Yes

Crowding Pale optic discs optic Pale

No Yes No Yes No Yes No Yes No No Yes No No No Yes No Yes Yes No

ystagmus N

No Yes No Yes No Yes Yes No No No No No No No No No Yes No

disorder

ye movement movement ye E

No No No No No Yes Yes No No Yes Yes No Yes No No Yes Yes No

trabismus S

Yes Yes No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Visual field defects field Visual

Yes No No Yes Yes No Yes Yes No Yes No No No Yes

S D O acuity Visual

LP 0.1 0.3 0.3 0.3 0.12 0.2 0.08 0.13 0.03 LP 0.1 0.06 0.1 0.2 0.2 0.15 0.05 0.1

OS error efraction R

S-6.00 S+3.00=C-0.50x180” S+4.75=C-1.00x140” Emmetropia S+1.00 S-10.00 C-4.00 S-3.00 S-2.25 S+3.00=C-1.00x90” C-2.00x120”

D O

efraction error error efraction R

S-6.00 S+3.00 S+4.50=C- 3.25x180” S+2.00=C-1.00x0” S+1.00 S-9.50=C-2.00x10” C-4.50 S-3.00 S-1.50=C-1.50x45” S+3.00=C-1.00x90” C-2.00x60°

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

35 m 13 y 8 y 12 y 28 m 9 y 13 y 11 y 8 y 38 m 18 y 9 y 9 m 46 m 10 y 4 y 13 y 9 y 8 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 422 421 420 417 416 414 413 412 411 410 408 409 407 403 402 401 398 397 395 A

Low vision due to CVI: differentiating between acquired and genetic causes

performances

luctuating visual visual luctuating F

Yes Yes Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A Yes

Chapter 2.1 the target the

ooking away from from away ooking L

Yes Yes

taring at light at taring S Crowding

Yes Yes Pale optic discs optic Pale

No No Yes No No No No No Yes No No No Yes Yes No No

ystagmus N

No Yes No No Yes Yes No No No No No No No Yes No

disorder

ye movement movement ye E

No Yes Yes No No No Yes Yes No Yes No No No No Yes No

trabismus S

No Yes Yes Yes No Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Visual field defects field Visual

No No Yes No No No Yes No No Yes No No No Yes No

S D O acuity Visual

0.1 0.25 0.1 0.2 0.2 0.06 0.125 0.07 0.06 0.08 0.04 0.3 0.2 0.25 0.03 0.25

OS error efraction R

S-5.50=C-0.50x118 S+1.50=C-1.50x180” S+4.50 S+0.50=C-1.00x180” S+1.50 S+3.00=C- 3.00x180” Emmetropia S+1.50=C-0.75x7” S+3.00 S+1.50=C-2.50x35”

D O

efraction error error efraction R

S-5.25=C-0.75x73” S+1.50=C- 2.50x180” S+4.50=C- 0.50x180” S+0.50 S+2.00=C-0.50x90” S+3.00 S+1.00=C- 1.00x180” C-0.25x160” S+4.00 S+1.00=C- 1.00x180”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

12 y 6 y 9 y 12 y 11 y 7 y 47 m 12 y 15 m 14 y 8 y 4 y 43 m 5 y 24 m 7 y ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 450 448 447 445 443 441 440 438 433 432 429 428 426 425 424 423 A

Chapter 2.1

performances

luctuating visual visual luctuating F

Yes Yes

abnormalities

ixatation ixatation F

Yes Yes Yes Yes

recognition

object/ face face object/

Problems with with Problems

dominates

uditive stimuli stimuli uditive A

the target the

ooking away from from away ooking L

Yes Yes

taring at light at taring S

Yes Yes Crowding

Yes Pale optic discs optic Pale

Yes Yes Yes Yes Yes No

ystagmus N

No Yes No No No

disorder

ye movement movement ye E

No Yes No No Yes No

trabismus S

Yes Yes Yes Yes Yes Yes Visual field defects field Visual

Yes Yes Yes Yes Yes

S D O acuity Visual

0.25 0.025 0.07 0.2 0.07 0.2

OS error efraction R

S+5.50=C-0.75x35” S+1.00 S-1.00=C-1.00x35” S+2.00=C-1.50x170

D O

efraction error error efraction R

S+5.50 S+2.00 S-1.50=C-2.00x35” S+1.50=C-0.75x75”

(m), years (y)) years (m),

amination (months (months amination

- ex ophtalmological

ge most recent recent most ge A

28 m 7 y 36 m 10 y 6 y 30 m ID dditional file 1: Phenotype information of all patients involved, part 2 (continued) involved, of all patients dditional file 1: Phenotype information 458 457 456 454 452 451 A perception. LP = Light

90 Low vision due to CVI: differentiating between acquired and genetic causes

Additional file 2: Ocular findings in individuals with a normal or abnormal MRI scan of the brain Raw Normal MRI (N=28) Abnormal MRI (N=178) p-value Mean age most recent examination 76 (SD 70) 61 (SD 48) 0.364 (months) Affected N/ Available N Affected N/ Available N Men 17/28 (61%) 94/178 (53%) 0.542 Vigabatrin use 4/28 (14%) 30/178 (17%) 1.000 Myopia <-4 0/28 (0%) 5/178 (3%) 1.000 Chapter 2.1 Hypermetropia >+4 3/28 (11%) 16/178 (9%) 0.728 Visual acuity <0.05 or <1.6 cycles/ 5/28 (18%) 41/178 (23%) 0.663 cm at 55 cm Strabismus 18/27 (67%) 136/171 (80%) 0.141 Nystagmus 6/26 (23%) 73/155 (47%) 0.031 Visual field defect 11/22 (50%) 91/146 (62%) 0.349 Hemianopia 3/11 16/91 - Upper or lower visual field defect 4/11 20/91 - Constriction of visual field 4/11 55/91 - (Partial) pale optic disc 4/28 (14%) 82/169 (49%) 0.001* *p-Values in bold represent differences that passed the Benjamini–Hochberg criterion.

2.2

Chromosomal aberrations in cerebral visual impairment

Daniëlle G.M. Bosch, F. Nienke Boonstra, Margot R.F. Reijnders, Rolph Pfundt, Frans P.M. Cremers, Bert B.A. de Vries.

Eur J Paediatr Neurol 2014; 18: 677-684. Chapter 2.2

Abstract

Background Cerebral visual impairment (CVI) is a disorder in projection and/or interpretation of the visual input in the brain and accounts for 27% of the visually impaired children. Aim A large cohort of patients with CVI was investigated in order to ascertain the relevance of chromosomal aberrations in the aetiology of this disorder. Methods 607 patients with CVI and a visual acuity ≤0.3 were assessed for the presence of a chromosomal aberration retrospectively. The observed aberrations were classified for pathogenicity. Results A total of 98 chromosomal aberrations were found in 79 persons (13%) of the cohort. In nine persons it was not possible to classify the clinical implication of the aberration, due to lack of detailed information. In 70 persons it was possible to classify the aberration for causality: in 41 patients the aberration was associated with CVI, in 16 it was unknown and in 13 the aberration was unlikely to be associated with CVI. For four aberrations, present in 26 patients, the association with CVI has been reported before: trisomy 21, 1p36 deletion syndrome, 17p13.3 deletion syndrome (Miller-Dieker syndrome) and 22q13.3 deletion syndrome (Phelan-McDermid syndrome). The chromosomal aberrations in another 15 patients were for the first time associated with CVI. Conclusions Chromosomal aberrations associated with CVI were found in 7% (41/607) of patients, of which 37% (15/41) have not been reported before in association with CVI. Therefore, in patients with CVI chromosomal investigations should be routinely performed to warrant a good clinical diagnosis and counselling.

94 Chromosomal aberrations in CVI

Introduction

Cerebral visual impairment (CVI) is one of the major causes of visual impairment in the developed world, as it accounts for 27% of low vision in childhood.53,55 It includes all visual dysfunctions caused by damage to, or malfunctioning of, the retrochiasmatic pathways in the absence of damage to the anterior visual pathways or any major ocular disease.49,51 CVI is diagnosed when no ocular abnormality can explain the impairment in vision. This impairment can consist of a reduced visual acuity, and/or visual field defects.47 Furthermore, there can be an abnormal visual behaviour, such as staring into light or delayed fixation. Deficits in higher perceptual functions, for example difficulties with recognition of objects and faces, or visiospatial disorders can occur, and are sometimes the only features of CVI.46,50,85 The prevalence of persons with CVI without a severe visual acuity loss or visual field defects is unknown. An important cause of CVI is acquired damage to the brain, mainly the result of perinatal Chapter 2.2 problems, such as cerebral haemorrhage or periventricular leukomalacia. Cerebral damage during the prenatal (e.g. congenital CMV infection), neonatal (e.g. hypoglycaemia) and childhood (e.g. meningitis or head trauma) period are less frequent causes of CVI.47 Furthermore, West syndrome and hydrocephalus can result in CVI.115,116,223 So far, less attention has been paid to genetic causes of CVI, although several neurodegenerative causes have been described.62 CVI is often part of a more complex phenotype, consisting of intellectual disability, epilepsy and/or deafness.51,79,224 Recently, the awareness is growing that CVI is common in intellectual disability and in a large cohort of 923 patients with intellectual disability visual impairment due to CVI was present in 5%.225 In patients with intellectual disability a causative chromosomal aberration can be found in about 5-30%, depending on the resolution of the technique used and the cohort selected.138,144,152,226,227 Since 1970 karyotype analysis has been used to detect chromosomal aberrations larger than 5 Mb, but since the introduction of array-based comparative genomic hybridization (array CGH) techniques around ten years ago, the resolution increased significantly allowing the detection of causative aberrations as small as 20 kb. Besides intellectual disability also other neurocognitive disorders, such as autism and schizophrenia, can be caused by chromosomal aberrations.228 Moreover, recently, deletions of 5q15 including the NR2F1-gene were described to cause CVI.191 This initiated our study to ascertain to what extend chromosomal aberrations can be the underlying cause of CVI. Here we provide an overview of the chromosomal aberrations found in the largest cohort of patients with CVI described so far.

95 Chapter 2.2

Methods

All patients diagnosed with CVI were seen and tested in Bartiméus, an institute for diagnostics, rehabilitation and schooling of the visually impaired in the Netherlands. CVI was diagnosed by a paediatric ophthalmologist under the following criteria: no other ocular diagnosis which could explain the visual impairment or visual field defect, and/or typical features such as poor fixation or crowding, and/or CVI found at neuropsychological investigation. Neuropsycho- logical investigation was, however, not possible in a majority of the individuals, because of their developmental age. The individuals with CVI included in this study underwent their first ophthalmological examinations between 1-1-1993 and 1-1-2013. Additional inclusion criteria were low vision, defined as a visual acuity of ≤0.3 or <9.8 cycles/cm at 55 cm or a visual field of 30° or below.53 Under the age of three the visual acuity was measured by Teller Acuity Cards and defined as decreased, when the cycles/degree were below the standard deviation reported by Courage and Adams.196 Visual acuity in young children or in individuals with a young developmental age was measured by preferential looking (Teller Acuity Cards, 55 cm), Cardiff cards (1 m), picture optotypes (mainly LH Symbols at 3 m), or number optotypes (6 m). The confrontational method with white Stycar balls, 5 cm in diameter, was used to estimate the visual field. Exclusion criterion was a second ocular diagnosis causing low vision, such as cataract, retinopathy of prematurity, congenital nystagmus, or primary (hereditary) optic atrophy. All medical correspondence of the patients with CVI was screened for chromosomal investigations. For all patients with a chromosomal aberration the available clinical data were screened for risk factors for CVI, which are perinatal problems, periventricular leukomalacia, stroke, West syndrome, hydrocephalus, hypoglycaemia or a gestational age <37 weeks. In addition, the results of the most recent ophthalmologic examination were collected, including binocular visual acuity, visual fields, strabismus, nystagmus, refraction error and the appearance of the optic disc. All chromosomal aberrations were classified for pathogenicity for CVI according to an adapted workflow of Koolen et al. (Figure 1).144 The database of genomic variants (http://dgv.tcag.ca/dgv/app/home) was used to filter against common variants. When no cerebral damage or other risk factor was found to explain CVI it was assumed that the chromosomal aberration was possibly associated with CVI.

Results

Of the 607 individuals with CVI and a visual acuity of ≤0.3, 197 (32%) had undergone chromosomal investigations, either karyotype analysis (n=109 ) or array CGH (n=75). In 13 patients the method was not specified. In 79 patients (79/607, 13%) 98 aberrations were found (Supplementary Table 1). The phenotype of these patients is described in Supplementary Table 2. Three patients (patients 12, 41 and 45) have been reported previously.229-231 For ten

96 Chromosomal aberrations in CVI

Chromosomal aberration n=84

Yes Common chromosomal aberration? n=1 1) Internal dataset of normal controls 2) Publically available datasets of normal controls

No n=83 Yes n=26 CVI previously reported?

No n=57 No n=15 Chapter 2.2 Segregates with the phenotype?

Yes/ unknown n=42 No n=20 Risk factors for CVI* or unknown birth term?

Yes n=22

Associated with CVI Unknown whether Unlikely to be associated associated with CVI with CVI n=46 n=22 n=16

Figure 1: Workflow for the decision making of chromosomal aberrations in individuals with cerebral visual impairment. *These risk factors are a gestational age <37 weeks, hypoglycaemia, hydrocephalus, meningitis/encephalitis, perinatal problems, periventricular leukomalacia, stroke and West syndrome.

aberrations it was not possible to obtain the exact breakpoints of the aberration. In addition, the clinical interpretation proved to be difficult for four aberrations. First, a del(22)(p11.1) deletion, patient 1, this region contains no genes and was therefore not classified. Secondly, a small duplication dup(6)(p25.3p25.2) in patient 47 was of unknown inheritance. This gain contains partly the GMDS-gene, GDP-mannose 4,6-dehydratase. It is unknown whether this duplication disrupts the GMDS-gene, therefore the duplication could not be classified. Lastly, two duplications on chromosome X in two girls, patient 59 and 60, were not classified, because detailed phenotype information and the X-inactivation profile was lacking. The remaining 84 aberrations, consisting of three structural aberrations without evidence for aneuploidy, 23 whole chromosome aberrations, and 58 partial chromosomal aberrations, were classified for causality according to the workflow (Figures 1 and 2).

97 Chapter 2.2

1 2 3 4 5 6 7 8 9 10 11 12

44 52 54 1 42 3 43 5 45* 48 51 64 55 7 53 11* 65 55 12 9

61*

66 13 46 46 49

10 10 8

50 46 62 63* 6

Associated with CVI Unknown whether associated with CVI 2 2 Unlikely to be associated with CVI

13 14 15 16 17 18 19 20 21 22 X Y

16 15 58 N 72 73 70

18 7 8 69 17 69 14 14 58 56 40

71 2

68 50

Figure 2: Overview of the chromosomal aberrations reported in patients with CVI. Copy number losses are depicted on the left-hand side and copy number gains on the right-hand side of the chromosomes by coloured bars. Underlined numbers indicate that more than one aberration was present in the patient. 11* mosaic triplication, 45* mosaic triplication, 61* triplication, 63* partially duplicated and partially triplicated. N: patients 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, and 39 had a trisomy 21, and 28 mosaic trisomy 21.

Unlikely to be associated with CVI 16 chromosomal aberrations were classified as unlikely to be associated with CVI (Figure 1). One aberration (patient 61) was reported several times in normal controls in the database of genomic variants. The other 15 aberrations did not segregate with the CVI phenotype, either inherited from an unaffected parent (n=11), not present in a similarly affected sib (n=2), or was not fitting with the phenotype reported in literature (n=2, patient 72 and 73). Patient 72 had a trisomy X. However, she was known with a pontocerebellar hypoplasia type 2, leading

98 Chromosomal aberrations in CVI to the conclusion that the triple X was a coincidental finding and probably not associated with CVI. In patient 73 a duplication of Y was present, which results in no or only a mild phenotype not related to CVI, and was therefore also considered as a coincidental finding, unlikely to be associated with CVI. Those 16 aberrations were present in 14 patients. However, patient 46 had three aberrations, of which one, a deletion del(6)(q14.1), was inherited and classified as unlikely to be associated, but the two other deletions del(5)(q14.3) and del(6) (q25.3), proved to be de novo and classified as unknown. In total, in 13 individuals all the aberrations found, were classified as unlikely to be associated with CVI.

Unknown whether associated with CVI 22 chromosomal aberrations were classified as unknown (Figure 1). The chromosomal aberrations were not present in a dataset of normal controls and did segregate with the phenotype, but there were other factors present that might have caused the CVI. These other factors con- Chapter 2.2 sist of West syndrome, hydrocephalus, perinatal problems, stroke, meningitis/encephalitis, and hypoglycaemia (n=13). In addition, when the gestational age was unknown (n=7) or <37 weeks (n=2) the aberration was classified as unknown. Those 22 aberrations were present in 17 patients. However, patient 14 had a duplication dup(15)(q15) of unknown causality and a deletion del(22)(q13.3), which is associated with CVI, resulting in 16 patients in which it was unknown whether the aberration found caused CVI.

Associated with CVI 46 chromosomal aberrations in 41 patients were associated with CVI (Figure 1, Table 1). CVI was previously reported in 26 patients and CVI was associated with the chromosomal aberration in another 15 patients. These aberrations were not present in a dataset of normal controls and segregated with the phenotype. Moreover, no other factors that could have caused CVI were present and the patients had a normal gestational age, rendering the chromosomal aberrations likely to be associated with CVI.

99 Chapter 2.2

Table 1: Description of persons with a chromosomal aberration associated with CVI Pt Chromosomal aberration Male/ GA ID MRI brain report Epilepsy Other Female (weeks) Factor 1 del(1)(p36.3)* F 40 + + P 2 del(15)(q24.3q25) M 37 + Minor aspecific abnormalities - dup(2)(q37.3) occipital del(2)(q37.3) 3 del(4)(p15) F 40 + 4 del(4)(q28.3q31.1) M AT + Small area with low signal density left temporal in optic radiation, probably cavernous haemangioma. 5 del(5)(p13) M 37 + Normal - 6 del(5)(q35) F 40 + Small choroid plexus cyst - 7 del(9)(p), M 41 + + dup(13)(q) 8 dup(9)(q34), dup(15)(q13) M 42 + - 9 del(10)(p12.33p12) F 40 - Normal - 10 dup(11)(q23.3) M 40 + - del(12)(q24.3) 11 Tetrasomy 12p M 37 + + 12 dup(12)(p13.1q11) F 38 + Normal - 13 dup(12)(q21.1q21.2) M 40 + 14 dup(15)(q15) F + - del(22)(q13) 15 del(17)(p13.3)* F 43 + Lissencephaly + 16 del(17)(p13.3)* M 40 + Lissencephaly + 17 dup(17)(q) M 39 + 18 del(18)(q) M 38 + - 40 del(22)(q13.3)* F + Normal - G 41 Ring X,45X/46,X F 39 + - 19 Trisomy 21* M + - 20 Trisomy 21* M + - 21 Trisomy 21* M + 22 Trisomy 21* F + - 23 Trisomy 21* F 41 + + 24 Trisomy 21* M 39 + Normal + 25 Trisomy 21* M 40 + 26 Trisomy 21* M + - 27 Trisomy 21* M 39 + - 28 Trisomy 21*, mosaic M 39 + - 29 Trisomy 21* F 39 + 30 Trisomy 21* M 39 + - P

100 Chromosomal aberrations in CVI

Table 1: Description of persons with a chromosomal aberration associated with CVI (continued) Pt Chromosomal aberration Male/ GA ID MRI brain report Epilepsy Other Female (weeks) Factor 31 Trisomy 21* M 37 + 32 Trisomy 21* M 28 + - 33 Trisomy 21* M + 34 Trisomy 21* M + - 35 Trisomy 21* M 40 - 36 Trisomy 21* M + 37 Trisomy 21* M 38 + - 38 Trisomy 21* M + - 39 Trisomy 21* M 32 + - AT=at term, G=hypoglycaemia, GA= gestational age, ID=intellectual disability, P=perinatal problems. *Indi- cates that CVI has been reported previously in this aberration. Chapter 2.2

Discussion

In our cohort of 607 patients with CVI only 32% had a reported chromosome testing, which probably reflects an underestimation as normal results may not always have been registered properly. In 13% a chromosomal aberration was found, mainly by classical karyotyping which has a lower resolution than novel array CGH techniques (5 Mb versus down to 20 kb). After classification, the chromosomal aberrations were considered associated with CVI in 7% (41/607), unknown in 3% (16/607), and unlikely to be associated with CVI in 2% (13/607) of the patients. All patients with a chromosomal aberration in this cohort were intellectually disabled. The association between the severity of the intellectual disability and a higher risk for low vision has been reported before, however, the majority of individuals with intellectual disability do have normal visual acuity.225

Aberrations with known associations with CVI In four of the aberrations, CVI has been reported previously, even though in medical literature results of ophthalmological examination, especially visual acuity measurement or assessment for CVI, are sparsely reported. The most often found aberration in our CVI cohort was trisomy 21 in 21 patients, which has a known association with CVI.130-132 A lower visual acuity in Down syndrome might be explained by accommodation problems, which is present in a majority of individuals with Down syndrome.131 Bifocals improve the near visual acuity (15-40 cm), but not the distance visual acuity (3 m).232,233 Whether poor accommodation played a role in the visual acuity measurement at 55cm in our cohort is unknown. Nevertheless, the low vision in Down syndrome patients cannot be completely attributed to poor accommodation or other ophthalmological abnormalities.130-132,234 Supporting evidence for visual cortex malfunction

101 Chapter 2.2 has been found by Vernier acuity measurements and steady state and sweep visual evoked potentials in combination with behavioural testing.130,234 In another two patients (patients 15 and 16) a deletion del(17)(p13.3) was present, causing Miller-Dieker syndrome. The main feature of this syndrome is lissencephaly and it is caused by the haploinsufficiency of the LIS1-gene.211,235 In a study by Nabi et al. in 20 patients with Miller-Dieker syndrome, one patient was diagnosed with CVI,211 making the lissencephaly associated with CVI in both patients. A deletion del(22)(q13.3), identified in two other patients (patients 14 and 40), leads to Phelan-McDermid syndrome. This syndrome presents with neonatal hypotonia, intellectual disability and dysmorphism.236 CVI has been observed in about 6% of the patients with this syndrome, but it is most likely still underdiagnosed.236 The last known syndromal association with CVI was the 1p36.3 deletion syndrome, present in patient 1. This clinically recognizable syndrome with intellectual disability and structural brain abnormalities is characterized by typical facial features, including straight eyebrows, deep set eyes, midface hypoplasia, broad and flat nasal root/bridge, long philtrum, and pointed chin.128,129 In literature CVI features like visual inattentiveness, such as absence of attentive visual behaviour with fixation and following movements are present in 30%-64% of the patients.128,129 However, visual acuity assessment was not mentioned in those reports.

Aberrations newly associated with CVI There were 15 patients with 20 chromosomal aberrations that have not been associated with CVI so far. In seven patients these aberrations caused well-know OMIM (Online Mendelian Inheritance in Man) annotated syndromes (http://www.ncbi.nlm.nih.gov/omim) and those syndromes will be discussed more extensively. The first OMIM-annotated syndrome is Wolf-Hirschhorn syndrome (OMIM #194190), caused by 4p-deletion. This recognizable syndrome features intellectual disability, microcephaly, a Greek warrior helmet appearance of the nose, epilepsy and growth delay.237 In our cohort a 4p-deletion was identified in patients 3, 44 and 45. For patient 44 and 45 it remained unknown whether the chromosomal aberration was related to CVI, because in patient 44 the gestational age was unknown and patient 45 had West syndrome. In patient 3, however, there was no other possible explanation for CVI. In literature 20 patients with Wolf-Hirschhorn syndrome have been reported, who underwent investigation for ocular abnormalities. However, the visual acuity nor assessment for cerebral visual impairment were reported.238,239 The 9p-deletion syndrome (OMIM #158170) was identified in four persons. This syndrome is characterized by intellectual disability, hypotonia, dysmorphic facial features, such as trigono- cephaly, midface hypoplasia, and a long philtrum.240,241 For one patient (patient 7) the deletion was associated with CVI, whereas for the three others (patients 54, 55 and 65) other possible contributive factors might have played a role. There are several reports of patients with a 9p- deletion, however, ophthalmological examination has not been described.240,241

102 Chromosomal aberrations in CVI

A 5p deletion leading to cri du chat syndrome (OMIM #123450) was identified in a single patient (patient 5). The key feature of this syndrome is a typical high-pitch cat-like cry, and is present when the deletion comprises the cytogenetic band “5p15.3”. Patients with a deletion comprising the cytogenetic band “5p15.2” have distinct facial dysmophisms, strabismus, microcephaly, pre- and postnatal growth deficiency, and intellectual disability.242,243 The ocular phenotype may consist of divergent strabismus, myopia and cataract. Another OMIM-annotated syndrome identified in patient 11: a mosaic tetrasomy 12p, causing the Pallister-Killian syndrome (OMIM #601803). Patients with this syndrome have intellectual disability, pigmentary skin abnormalities, congenital malformations, and craniofacial dysmorphisms, including bitemporal alopecia and hypertelorism.244 In Pallister-Killian syndrome several ocular disorders have been described, such as strabismus, nystagmus, cataract and optic nerve atrophy but no CVI, so far.245 A deletion on chromosome 5, del(5)(q35), including the NSD1-gene, was identified in patient Chapter 2.2 6. Deletion of and mutations in this gene cause Sotos syndrome (OMIM #117550), that is characterized by overgrowth resulting in tall stature and macrocephaly. Furthermore, learning disability and a characteristic facial appearance consisting of a high broad forehead and a pointed chin are present in more than 90% of the affected.246 So far, no large scale ophthalmological study in a cohort of Sotos patients has been performed, but astigmatism, myopia, cataract, delayed visual maturation, and nystagmus have been described.246 One patient (patient 2) had three aberrations, a deletion on chromosome 2 del(2)(q37.3), a duplication on chromosome 2dup(2)(q37.3) and a deletion on chromosome 15 del(15) (q24.3q26). The latter deletion comprises the cytogenetic bands 15q25.2 and 15q25.3 which have been reported to cause a rather variable phenotype (OMIM #614294).247 One reported patient inherited the deletion from a normal parent; however, ophthalmological examination is not described. A deletion on the long arm of chromosome 18, overlapping the 18q22.3q23 region, was identified in patient 18, which led to the De Grouchy syndrome (OMIM #601808). The features of this syndrome are intellectual disability, short stature, delayed myelination, congenital aural atresia, foot deformities, and dysmorphism including midface hypoplasia, hypertelorism and microcephaly.248 There is only one report of a girl with severe epilepsy, optic atrophy and visual loss.249 In summary, in our large cohort of patients with CVI, chromosomal aberrations are associated with CVI in 7%. Further, we were able to associate several chromosomal regions with CVI for the first time. Our study warrants chromosomal investigations in patients with CVI, because identifying the underlying cause is of great value for patients and his/her family and ensures the best clinical care for the patient. When more aberrations will be found in CVI this may further narrow down the candidate regions identified and may even lead to candidate genes which will eventually lead to an increased understanding of the development and function of the visual system.

103 Supplementary tables Breakpoints positionsBreakpoints in Mb ( S ize)* 239.95-241.37 (1.42 Mb) 241.44-243.00 (1.56 Mb) 32.64-33.32 (0.08 Mb) 138.01-140.74 (2.73 Mb) 159.46-160.01 (0.56 Mb) Method used Karyotype Affymetrix Affymetrix 250k Affymetrix Affymetrix 250k Affymetrix 500k Karyotype Cytoscan Cytoscan HD Agilent Agilent 180k FISH Karyotype ther factor that O cause CVImay + - - + + + + - - + + - - - + egregation/finding egregation/finding S with phenotype associated De novo Inherited Inherited De novo Inherited De novo Inherited De novo De novo CVI reported in (PMID) literature 18245432, 12687501 Present Present in DGV Yes Associated with CVI Associated Classification Classification CVI Associated with CVI Associated Associated with CVI Associated Unlikely to be Unlikely to with CVIassociated Unknown wether with CVIassociated Associated with CVI Associated Associated with CVI Associated Unknown wether with CVIassociated Unknown wether with CVIassociated Unlikely to be Unlikely to with CVIassociated Associated with CVI Associated Associated with CVI Associated Unknown wether with CVIassociated p36.3 Band q37.3 q37.3qter p22.3 q q p pterp15 q28.3q31.1 p p q32.2 pterp13 q35 p 1 2 2 2 2 2 3 4 4 4 4 4 5 5 5 Chromosome berration Deletion A Duplication Deletion Triplication Deletion Deletion Deletion Deletion Deletion Deletion Deletion Duplication Deletion Deletion Triplication Triplication mosaic 1 Patient 2 2 61 74 74 42 3 4 43 44 62 5 6 45 upplementary Table 1: Chromosomal aberrations in patients with CVI in patients aberrations 1: Chromosomal Table S upplementary

104 Chromosomal aberrations in CVI Breakpoints positionsBreakpoints in Mb ( S ize)* 88.46-91.69 (3.23 Mb) 158.57-158.93 (0.34 Mb) 80.75-81.09(0.34 Mb) 2.17-2.38 (0.21 Mb) 87.97-94.40 (6.43 Mb) 161.60-161.67 (0.73 Mb) 161.68-161.82 (0.14 Mb) 101.37-107.29 (5.91 Mb) 1.78-1.98 (0.20 Mb) Method used Agilent Agilent 180k Agilent Agilent 180k Agilent Agilent 180k Affymetrix 2.7M Karyotype Agilent Agilent 180k Cytoscan Cytoscan HD Cytoscan Cytoscan HD Agilent Agilent 180 k Karyotype Karyotype Agilent Agilent 180k Karyotype Karyotype ther factor that O cause CVImay + + + - + + - - + + + - - - - Chapter 2.2 egregation/finding egregation/finding S with phenotype associated De novo De novo Inherited De novo De novo Inherited Inherited De novo De novo Inherited De novo CVI reported in (PMID) literature Present Present in DGV Unknown wether with CVIassociated Classification Classification CVI Unknown wether with CVIassociated Unlikely to be Unlikely to with CVIassociated Unknown wether with CVIassociated Unknown wether with CVIassociated Unlikely to be Unlikely to with CVIassociated Unlikely to be Unlikely to with CVIassociated Unknown wether with CVIassociated Unknown wether with CVIassociated Unknown wether with CVIassociated Unlikely to be Unlikely to with CVIassociated Associated with CVI Associated Associated with CVI Associated q14.3 Band q25.3 q14.1 p25.3p25.2 pterp25.3 q15p16.1 q26 q26 q36.1q36.3 p p23p11.2 p23.3 p q34qter 5 6 6 6 6 6 6 6 7 8 8 8 8 9 9 Chromosome berration Deletion A Deletion Deletion Duplication Deletion Deletion Duplication Triplication Duplication Duplication Duplication Duplication Duplication Deletion Duplication 46 upplementary Table 1: Chromosomal aberrations in patients with CVI (continued) in patients aberrations 1: Chromosomal Table S upplementary Patient 46 46 47 48 49 63 63 50 51 52 64 75 7 8

105 Chapter 2.2 Breakpoints positionsBreakpoints in Mb ( S ize)* 0.04-13.37 (13.33 Mb) 13.38-44.91 (31.53 Mb) 6.57-6.69 (0.12 Mb) 75.31-75.56 (0.25 Mb) 18.59-27.76 ( 9.17 Mb) 75.66-75.80 (0.15 Mb) Method used Karyotype Affymetrix Affymetrix 250k Affymetrix Affymetrix 250k Cytoscan Cytoscan HD Cytoscan Cytoscan HD Karyotype Karyotype Karyotype Karyotype Cytoscan Cytoscan HD ther factor that O cause CVImay + + + + - - - + - - + - - - - egregation/finding egregation/finding S with phenotype associated De novo De novo De novo Not present in affected sib in affected Not present Inherited De novo Inherited CVI reported in (PMID) literature Present Present in DGV Unknown wether with CVIassociated Classification Classification CVI Unknown wether with CVIassociated Unknown wether with CVIassociated Unknown wether with CVIassociated Unlikely to be Unlikely to with CVIassociated Unlikely to be Unlikely to with CVIassociated Associated with CVI Associated Associated with CVI Associated Unlikely to be Unlikely to with CVIassociated Associated with CVI Associated Associated with CVI Associated Associated with CVI Associated Associated with CVI Associated p Band p p23 p23p12 p24.1 q21.13 p12.33p12.1 q23.3qter inv(11) (q21,22q23.3) q24.3qter p p13.1q11 q21.1q21.2 9 9 9 9 9 9 10 11 10 11 12 11 12 12 12 Chromosome berration Duplication A Deletion Deletion Duplication Deletion Deletion Deletion Duplication Inversion Deletion Tetrasomy Tetrasomy mosaic Duplication Duplication 53 upplementary Table 1: Chromosomal aberrations in patients with CVI (continued) in patients aberrations 1: Chromosomal Table S upplementary Patient 54 55 55 65 66 9 76 10 67 77 10 11 12 13

106 Chromosomal aberrations in CVI Breakpoints positionsBreakpoints in Mb ( S ize)* 94.47-94.49 (0.02 Mb) 151.56-159.13 (7.57 Mb) 77.77-102.37 (24.60 Mb) 28.12-28.41 (0.29 Mb) 6.25-6.53 (0.28 Mb) 72.03-72.09 (0.06 Mb) 43.74-44.20 (0.46 Mb) Method used Karyotype BAC array BAC Agilent Agilent 180 k Affymetrix Affymetrix 250k Karyotype Karyotype Array CGH Array Cytoscan Cytoscan HD Cytoscan Cytoscan HD Affymetrix Affymetrix 250k ther factor that + O cause CVImay - - + - - - + - - - + - - - + Chapter 2.2 egregation/finding egregation/finding S with phenotype associated Inherited De novo De novo De novo Inherited Inherited Not present in affected sib in affected Not present De novo CVI reported in (PMID) literature 12825057 12825057 Present Present in DGV Associated with CVI Associated Classification Classification CVI Unlikely to be Unlikely to with CVIassociated Unknown wether with CVIassociated Associated with CVI Associated Associated with CVI Associated Unknown wether with CVIassociated Unlikely to be Unlikely to with CVIassociated Unlikely to be Unlikely to with CVIassociated Unlikely to be Unlikely to with CVIassociated Associated with CVI Associated Associated with CVI Associated Associated with CVI Associated Unknown wether with CVIassociated q Band q31.3 q32.31q32.33 q24.3q26 q13qter q15qter q13.1 p13.3 q22.2 p13.3 p13.3 q q21.31 13 12 13 14 14 15 15 15 15 15 16 16 17 17 17 17 Chromosome berration Duplication A Deletion Deletion Duplication Deletion Duplication Duplication Duplication Deletion Duplication Deletion Deletion Deletion Duplication Deletion 76 7 upplementary Table 1: Chromosomal aberrations in patients with CVI (continued) in patients aberrations 1: Chromosomal Table S upplementary Patient 68 50 75 2 8 14 69 78 70 71 15 16 17 56

107 Chapter 2.2 Breakpoints positionsBreakpoints in Mb ( S ize)* 0.07-13.02 (12,95 Mb) Method used Karyotype Agilent Agilent 180k Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype ther factor that O cause CVImay + - + + + + + + - - - + - - - + - + + + egregation/finding egregation/finding S with phenotype associated De novo De novo CVI reported in (PMID) literature 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 18824732 Present Present in DGV Unknown wether with CVIassociated Classification Classification CVI Associated with CVI Associated Unknown wether with CVIassociated Unknown wether with CVIassociated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated t(1;17) (q21;p13.3) Band q p11.32p11.21 q13.33qter 17 18 18 20 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 Chromosome berration Translocatie A Deletion Duplication Duplication Trisomy Trisomy Trisomy Trisomy Trisomy Trisomy Trisomy Trisomy Trisomy Trisomy mosaic Trisomy Trisomy Trisomy Trisomy Trisomy Trisomy 57 upplementary Table 1: Chromosomal aberrations in patients with CVI (continued) in patients aberrations 1: Chromosomal Table S upplementary Patient 18 58 48 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

108 Chromosomal aberrations in CVI Breakpoints positionsBreakpoints in Mb ( S ize)* 39.03-48.08 (9.06 Mb) 43.51-43.88 (0.37 Mb) 73.71-78.03 (4.32 Mb) Karyotype Karyotype Karyotype Karyotype Karyotype Method used Agilent Agilent 180k Karyotype Karyotype Karyotype Array CGH Array Karyotype Karyotype Karyotype Karyotype 105k oligo array ther factor that - + - + + O cause CVImay + + - + + + - + - - - + Chapter 2.2 egregation/finding egregation/finding S with phenotype associated Inherited Not fitting with literature Not fitting with literature De novo De novo 18824732 18824732 18824732 18824732 18824732 CVI reported in (PMID) literature 2670140 2670140 Present Present in DGV Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated with CVI Associated Unknown wether with CVIassociated Classification Classification CVI Associated with CVI Associated Associated with CVI Associated Unlikely to be Unlikely to with CVIassociated Unlikely to be Unlikely to with CVIassociated Unlikely to be Unlikely to with CVIassociated Associated with CVI Associated q22.13q22.3 Band pterp11.1 q13qter q13.3qter q13.2 X,45X/46,X q11.2q22.3 q13.2q21.1 Y X X X X 21 21 21 21 21 21 22 22 22 22 22 22 Chromosome berration Trisomy Trisomy Trisomy Trisomy Trisomy Deletion A Deletion Deletion Deletion Deletion Ring Duplication Trisomy Duplication Duplication 35 36 37 38 39 58 upplementary Table 1: Chromosomal aberrations in patients with CVI (continued) in patients aberrations 1: Chromosomal Table S upplementary Patient 1 14 40 69 77 79 41 73 72 59 60 DGV=Database of Genomic Variants. *Genome build February 2009, Hg 19. *Genome build February Variants. of GenomicDGV=Database

109 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration Patient 1 2 3 4 5 6 7 8 9 Male/ Female F M F M M F M M F Gestational age (weeks) 40 37 40 AT 37 40 41 42 40 Birth weight (gram) 2200 3325 2080 2650 2100 2170 3800 3340 2500 Intellectual disability + + + + + + + + - Developmental age at 9 months at 2+3 years 3+1 years at 5 years IQ 77-89 calender age MRI cerebrum Minor aspecific Small area with Normal Small choroid plexus Normal abnormalities (based on abnormalities occipital low signal density cyst reports) left temporal in optic radiation, probably cavernous haemangioma. Epilepsy + - - - + - - Hearing impairment ------+ - Other Factor P Age at investigation 4 3+11 16 36 6 4 9 months 10 33 (years) Refraction OD S+3.00=C-1.75x160” S+5.50=C-2.00x180” S+6.25=C-0.50x131” S+1.00 S+8.50 S+2.00=C-5.00x180” S-0.50=C-0.50x180” S-0.50=C-1.25x100” Refraction OS S+3.00=C-1.75x175” S+5.50=C-2.00x180” S+6.25=C-0.50x131” S-1.00 S+9.00 S+1.00=C-4.00x180” S-0.75 S-1.75=C-1.75x90” Correction - - - + - + - - Vision 20/94 (0.2) 2/15 (0.13) 20/190 (0.10) 5/25 (0.2) 20/130 (0.15) 20/130 (0.15) LP 6/24 (0.25) 5/38 (0.13) Method TAC (55 cm) LH (2 m) TAC (55 cm) Landolt C 5 m TAC (55 cm) TAC (55 cm) Numbers (6 m) Landolt C (5 m) Visual field defects + - - + - + + Strabismus + - + + + + + + - Nystagmus + - - - - + - - - (Partial) optic disc pallor - - - + - - - - - Fluctuating visual + + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

110 Chromosomal aberrations in CVI

Epilepsy + - - - + - - Chapter 2.2 Hearing impairment ------+ - Other Factor P Age at investigation 4 3+11 16 36 6 4 9 months 10 33 (years) Refraction OD S+3.00=C-1.75x160” S+5.50=C-2.00x180” S+6.25=C-0.50x131” S+1.00 S+8.50 S+2.00=C-5.00x180” S-0.50=C-0.50x180” S-0.50=C-1.25x100” Refraction OS S+3.00=C-1.75x175” S+5.50=C-2.00x180” S+6.25=C-0.50x131” S-1.00 S+9.00 S+1.00=C-4.00x180” S-0.75 S-1.75=C-1.75x90” Correction - - - + - + - - Vision 20/94 (0.2) 2/15 (0.13) 20/190 (0.10) 5/25 (0.2) 20/130 (0.15) 20/130 (0.15) LP 6/24 (0.25) 5/38 (0.13) Method TAC (55 cm) LH (2 m) TAC (55 cm) Landolt C 5 m TAC (55 cm) TAC (55 cm) Numbers (6 m) Landolt C (5 m) Visual field defects + - - + - + + Strabismus + - + + + + + + - Nystagmus + - - - - + - - - (Partial) optic disc pallor - - - + - - - - - Fluctuating visual + + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

111 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 10 11 12 13 14 15 16 17 18 Male/ Female M M F M F F M M M Gestational age (weeks) 40 37 38 40 43 40 39 38 Birth weight (gram) 3000 3500 3205 2860 3425 2750 Intellectual disability + + + + + + + + + Developmental age at 2 years at 8 years 12-15 months at 17 years 1+2 years at calender age 1+8 years MRI cerebrum Normal Lissencephaly Lissencephaly abnormalities (based on reports) Epilepsy - + - + - + + - Hearing impairment ------+ Other Factor Age at investigation 8 8 16 17 12 5 7 7 8 (years) Refraction OD S+3.50 S+4.50=C-2.50x170” S-6.00 S+2.00=C-0.50x90” S+5.00=C-2.00x180” S+1.00 S+3.00=C-1.00x180” S+1.00=C-1.00x180” Refraction OS S+2.50 S+3.00=C-1.25x30” S-6.00 S+1.50 S+5.00=C-2.00x180” S+3.00 S+3.5=C-0.50x180” S+1.75=C-0.75x180” Correction ------Vision 20/190 (0.10) 20/150 (0.13) 20/63 (0.3) 20/260 (0.08) 20/300 (0.07) 20/150 (0.13) 20/1900 (0.01) 20/190 (0.10) 20/200 (0.10) Method TAC (55 cm) TAC (38 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (38 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - - + - + - - Strabismus + + + + + + + + + Nystagmus + + - - - - - + (Partial) optic disc pallor - - - + - - - - - Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

112 Chromosomal aberrations in CVI

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 10 11 12 13 14 15 16 17 18 Male/ Female M M F M F F M M M Gestational age (weeks) 40 37 38 40 43 40 39 38 Birth weight (gram) 3000 3500 3205 2860 3425 2750 Intellectual disability + + + + + + + + + Developmental age at 2 years at 8 years 12-15 months at 17 years 1+2 years at calender age 1+8 years MRI cerebrum Normal Lissencephaly Lissencephaly abnormalities (based on reports) Epilepsy - + - + - + + - Hearing impairment ------+ Other Factor Chapter 2.2 Age at investigation 8 8 16 17 12 5 7 7 8 (years) Refraction OD S+3.50 S+4.50=C-2.50x170” S-6.00 S+2.00=C-0.50x90” S+5.00=C-2.00x180” S+1.00 S+3.00=C-1.00x180” S+1.00=C-1.00x180” Refraction OS S+2.50 S+3.00=C-1.25x30” S-6.00 S+1.50 S+5.00=C-2.00x180” S+3.00 S+3.5=C-0.50x180” S+1.75=C-0.75x180” Correction ------Vision 20/190 (0.10) 20/150 (0.13) 20/63 (0.3) 20/260 (0.08) 20/300 (0.07) 20/150 (0.13) 20/1900 (0.01) 20/190 (0.10) 20/200 (0.10) Method TAC (55 cm) TAC (38 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (38 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - - + - + - - Strabismus + + + + + + + + + Nystagmus + + - - - - - + (Partial) optic disc pallor - - - + - - - - - Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

113 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 19 20 21 22 23 24 25 26 27 Male/ Female M M M F F M M M M Gestational age (weeks) 41 39 40 39 Birth weight (gram) 2575 3415 3500 2800 Intellectual disability + + + + + + + + + Developmental age at 7 months at 3+4 years 2 months at 10 months 1+6 years at calender age 3+10 years MRI cerebrum Normal abnormalities (based on reports) Epilepsy - - - + + - - Hearing impairment - + + - Other Factor Age at investigation 4 8 5 18 5 7 months 7 6 7 (years) Refraction OD S+3.00 S+3.50=C-1.50x180” S+0.25=C-2.00x172” S-6.00 S+4.75=C-0.50x180” S+2.50=C-1.50x180” S+6.00=C-1.50x90” S+3.00=C-1.75x40” S+3.75=C-0.25x90” Refraction OS S+3.00 S+3.00=C-1x180” S-1.50=C-1.75x160” S-5.00 S+5.50=C-0.50x180” S+2.50=C-1.00x180” S+4.50 S+2.25=C-0.75x30” S+3.75 Correction - - - - + - - + - Vision 20/470 (0.04) 20/130 (0.15) 20/180 (0.11) 20/190 (0.10) 20/130 (0.15) LP 20/63 (0.3) 3/10 (0.3) 20/94 (0.2) Method TAC (55 cm) TAC (55 cm) TAC (38 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Amsterdam pictures (3m) TAC (55 cm) Visual field defects - - - + - - Strabismus + - - + - + - - - Nystagmus + - - + + + - + - (Partial) optic disc pallor ------Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

114 Chromosomal aberrations in CVI

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 19 20 21 22 23 24 25 26 27 Male/ Female M M M F F M M M M Gestational age (weeks) 41 39 40 39 Birth weight (gram) 2575 3415 3500 2800 Intellectual disability + + + + + + + + + Developmental age at 7 months at 3+4 years 2 months at 10 months 1+6 years at calender age 3+10 years MRI cerebrum Normal abnormalities (based on reports) Epilepsy - - - + + - - Hearing impairment - + + - Other Factor Chapter 2.2 Age at investigation 4 8 5 18 5 7 months 7 6 7 (years) Refraction OD S+3.00 S+3.50=C-1.50x180” S+0.25=C-2.00x172” S-6.00 S+4.75=C-0.50x180” S+2.50=C-1.50x180” S+6.00=C-1.50x90” S+3.00=C-1.75x40” S+3.75=C-0.25x90” Refraction OS S+3.00 S+3.00=C-1x180” S-1.50=C-1.75x160” S-5.00 S+5.50=C-0.50x180” S+2.50=C-1.00x180” S+4.50 S+2.25=C-0.75x30” S+3.75 Correction - - - - + - - + - Vision 20/470 (0.04) 20/130 (0.15) 20/180 (0.11) 20/190 (0.10) 20/130 (0.15) LP 20/63 (0.3) 3/10 (0.3) 20/94 (0.2) Method TAC (55 cm) TAC (55 cm) TAC (38 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Amsterdam pictures (3m) TAC (55 cm) Visual field defects - - - + - - Strabismus + - - + - + - - - Nystagmus + - - + + + - + - (Partial) optic disc pallor ------Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

115 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 28 29 30 31 32 33 34 35 36 Male/ Female M F M M M M M M M Gestational age (weeks) 39 39 39 37 28 40 Birth weight (gram) 2800 4012 2880 3250 Intellectual disability + + + + + + + + Developmental age at 12-15 months at 12 3+6 years at calender age years 18 years MRI cerebrum abnormalities (based on reports) Epilepsy - - - - - Hearing impairment - + - - - - Other Factor P Age at investigation 7 12 10 27 6 16 10 4 7 (years) Refraction OD S+1.75 S+3.00=C-0.50x180” S-10.00 S+1.50=C-1.25x102” S+3.50=C-2.50x180” S+3.00=C-0.50x90” S+3.00=C-1.00x180” S+2.00 Refraction OS S+1.75 S+3.00 S-5.00 S+2.25=C-1.00x12” S+3.75=C-2.00x180” S+3.50=C-0.50x90” S+3.00=C-1.00x180” S+1.00 Correction - - + - - - + - Vision 20/63 (0.3) 20/94 (0.2) 20/190 (0.10) 20/130 (0.15) 20/94 (0.2) 6/19 (0.3) 20/94 (0.2) 20/190 (0.10) 3/9.5 (0.3) Method TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Cardiff (1 m) TAC (55 cm) TAC (55 cm) LH (3 m) Visual field defects - - - + - - - - - Strabismus - + + + + - + - + Nystagmus - - - + + + - + - (Partial) optic disc pallor - + - - - - - + - Fluctuating visual performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

116 Chromosomal aberrations in CVI

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 28 29 30 31 32 33 34 35 36 Male/ Female M F M M M M M M M Gestational age (weeks) 39 39 39 37 28 40 Birth weight (gram) 2800 4012 2880 3250 Intellectual disability + + + + + + + + Developmental age at 12-15 months at 12 3+6 years at calender age years 18 years MRI cerebrum abnormalities (based on reports) Epilepsy - - - - - Hearing impairment - + - - - - Other Factor P Chapter 2.2 Age at investigation 7 12 10 27 6 16 10 4 7 (years) Refraction OD S+1.75 S+3.00=C-0.50x180” S-10.00 S+1.50=C-1.25x102” S+3.50=C-2.50x180” S+3.00=C-0.50x90” S+3.00=C-1.00x180” S+2.00 Refraction OS S+1.75 S+3.00 S-5.00 S+2.25=C-1.00x12” S+3.75=C-2.00x180” S+3.50=C-0.50x90” S+3.00=C-1.00x180” S+1.00 Correction - - + - - - + - Vision 20/63 (0.3) 20/94 (0.2) 20/190 (0.10) 20/130 (0.15) 20/94 (0.2) 6/19 (0.3) 20/94 (0.2) 20/190 (0.10) 3/9.5 (0.3) Method TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Cardiff (1 m) TAC (55 cm) TAC (55 cm) LH (3 m) Visual field defects - - - + - - - - - Strabismus - + + + + - + - + Nystagmus - - - + + + - + - (Partial) optic disc pallor - + - - - - - + - Fluctuating visual performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

117 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 37 38 39 40 41 42 43 44 45 Male/ Female M M M F F F F F M Gestational age (weeks) 38 32 39 41 34 Birth weight (gram) 2800 1425 3390 2085 1970 2550 Intellectual disability + + + + + + + + + Developmental age at 7 months at 6 months at calender age 9 years 5 years MRI cerebrum Slightly enlarged abnormalities (based ventricles and slightly on reports) delayed myelination. Epilepsy ------+ + + Hearing impairment - - - - + + - Other Factor G M W Age at investigation 13 17 6 5 11 12 2+11 10 2 (years) Refraction OD S-2.00 S-9.00=C-1.50x62” S-0.50=C-0.25x180” S+3.00 S+2.00=C-3.00x180” S+4.00=C-2.25x20” Refraction OS S-3.00=C-1.00x180” Splan=C-2.00x180” S-8.00=C-1.00x115” S+0.75=C-1.00x180” S+3.00 S+4.50=C-2.00x180” S+3.00=C-2.50x180” Correction - - + - - - Vision 20/150 (0.13) 20/130 (0.15) 3/9.5 (0.3) 20/150 (0.13) 20/130 (0.15) 20/380 (0.05) 20/710 (0.03) 20/710 (0.03) LP Method TAC (38 cm) TAC (55 cm) LH (3 m) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - - - - - + + - Strabismus + + - + + - + + - Nystagmus ------+ - - (Partial) optic disc pallor ------Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

118 Chromosomal aberrations in CVI

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 37 38 39 40 41 42 43 44 45 Male/ Female M M M F F F F F M Gestational age (weeks) 38 32 39 41 34 Birth weight (gram) 2800 1425 3390 2085 1970 2550 Intellectual disability + + + + + + + + + Developmental age at 7 months at 6 months at calender age 9 years 5 years MRI cerebrum Slightly enlarged abnormalities (based ventricles and slightly on reports) delayed myelination. Epilepsy ------+ + + Hearing impairment - - - - + + - Other Factor G M W Chapter 2.2 Age at investigation 13 17 6 5 11 12 2+11 10 2 (years) Refraction OD S-2.00 S-9.00=C-1.50x62” S-0.50=C-0.25x180” S+3.00 S+2.00=C-3.00x180” S+4.00=C-2.25x20” Refraction OS S-3.00=C-1.00x180” Splan=C-2.00x180” S-8.00=C-1.00x115” S+0.75=C-1.00x180” S+3.00 S+4.50=C-2.00x180” S+3.00=C-2.50x180” Correction - - + - - - Vision 20/150 (0.13) 20/130 (0.15) 3/9.5 (0.3) 20/150 (0.13) 20/130 (0.15) 20/380 (0.05) 20/710 (0.03) 20/710 (0.03) LP Method TAC (38 cm) TAC (55 cm) LH (3 m) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - - - - - + + - Strabismus + + - + + - + + - Nystagmus ------+ - - (Partial) optic disc pallor ------Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

119 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 46 47 48 49 50 51 52 53 54 Male/ Female M M F F M F F F M Gestational age (weeks) 27 37 41 42 39 41 Birth weight (gram) 1280 3186 2800 2650 3800 3400 Intellectual disability + + + + + + + + Developmental age at 4 years at 10 years 1+4 years at 1+3 years at calender age 3+2 years 9 years MRI cerebrum Hypoplasia of Enlarged ventricles Small cerebellum Normal Ventral corpus callosum Hydrocephalus Hydrocephaly, delayed abnormalities (based corpus callosum and and periventricular agenesia and enlarged development of on reports) colpocephaly leukomalacia. ventricles. frontal lobes, delayed myelination Epilepsy - + + - + - - - + Hearing impairment + - - + Other Factor P W H H H G Age at investigation 1 1+3 10 4 5 5 9 5 4 (years) Refraction OD S+1.00=C-1.00x90” S+8.00 C-2.00x180” S+2.00=C-0.25x180” S- 15.00= C+8.00x110” S-1.50=C-2.50x15” Refraction OS S+1.00=C-1.00x90” S+8.00 S-2.50 S+2.50 S-14.00= C+6.50x90” S-2.00=C-2.25x150” Correction - - - + - Vision LP LP 20/94 (0.2) 20/130 (0.15) 20/63 (0.3) 20/80 (0.25) 20/80 (0.25) 20/1200 (0.02) LP Method TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - - + + + Strabismus + + - + - + + + + Nystagmus + + - - - - + + (Partial) optic disc pallor + + - - - + + + Fluctuating visual performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

120 Chromosomal aberrations in CVI

Other Factor P W H H H G Chapter 2.2 Age at investigation 1 1+3 10 4 5 5 9 5 4 (years) Refraction OD S+1.00=C-1.00x90” S+8.00 C-2.00x180” S+2.00=C-0.25x180” S- 15.00= C+8.00x110” S-1.50=C-2.50x15” Refraction OS S+1.00=C-1.00x90” S+8.00 S-2.50 S+2.50 S-14.00= C+6.50x90” S-2.00=C-2.25x150” Correction - - - + - Vision LP LP 20/94 (0.2) 20/130 (0.15) 20/63 (0.3) 20/80 (0.25) 20/80 (0.25) 20/1200 (0.02) LP Method TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - - + + + Strabismus + + - + - + + + + Nystagmus + + - - - - + + (Partial) optic disc pallor + + - - - + + + Fluctuating visual performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

121 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 55 56 57 58 59 60 61 62 63 Male/ Female F M M F F F M M M Gestational age (weeks) 40 38 39 40 32 37 38 Birth weight (gram) 2700 3515 1710 3710 2000 2795 4250 Intellectual disability + + + + + + + Developmental age at 6-10 years at calender age 16 years MRI cerebrum Hydrocephalus and Ischaemic damage Enlarged ventricles and Normal Delayed myelination, Normal abnormalities (based delayed myelination thalamus and left delayed myelination enlarged ventricles, on reports) occipital damage due to small cerebellum, mega abcess. cisterna magna. Epilepsy + + - + + - + - + Hearing impairment + - + + - - - - Other Factor W H,P M,S W Age at investigation 26 13 35 1+9 1+10 4 14 1+6 26 (years) Refraction OD S-3.25=C-1.25x3” S-5.00 S-10.00 S+1.75 S+2.00=C-1.00x180” Refraction OS S-1.00 S-3.00=C-1.00x170” S-2.00 S-9.00 S+1.75 S+2.00=C-1.00x180” Correction - - + + - - + - - Vision 20/190 (0.10) 20/130 (0.15) 6/18 (0.3) 20/260 (0.08) LP 20/130 (0.15) 20/130 (0.15) 20/130 (0.15) 5/20 (0.25) Method TAC (55 cm) TAC (55 cm) Numbers (6 m) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Landolt C (5 m) Visual field defects + - + + - + Strabismus + + + + + + - - Nystagmus + - - + - - + + (Partial) optic disc pallor - + + - + - + + + Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

122 Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 55 56 57 58 59 60 61 62 63 Male/ Female F M M F F F M M M Gestational age (weeks) 40 38 39 40 32 37 38 Birth weight (gram) 2700 3515 1710 3710 2000 2795 4250 Intellectual disability + + + + + + + Developmental age at 6-10 years at calender age 16 years MRI cerebrum Hydrocephalus and Ischaemic damage Enlarged ventricles and Normal Delayed myelination, Normal abnormalities (based delayed myelination thalamus and left delayed myelination enlarged ventricles, on reports) occipital damage due to small cerebellum, mega abcess. cisterna magna. Epilepsy + + - + + - + - + Hearing impairment + - + + - - - - Other Factor W H,P M,S W Age at investigation 26 13 35 1+9 1+10 4 14 1+6 26 (years) Refraction OD S-3.25=C-1.25x3” S-5.00 S-10.00 S+1.75 S+2.00=C-1.00x180” Refraction OS S-1.00 S-3.00=C-1.00x170” S-2.00 S-9.00 S+1.75 S+2.00=C-1.00x180” Correction - - + + - - + - - Vision 20/190 (0.10) 20/130 (0.15) 6/18 (0.3) 20/260 (0.08) LP 20/130 (0.15) 20/130 (0.15) 20/130 (0.15) 5/20 (0.25) Method TAC (55 cm) TAC (55 cm) Numbers (6 m) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Landolt C (5 m) Visual field defects + - + + - + Strabismus + + + + + + - - Nystagmus + - - + - - + + (Partial) optic disc pallor - + + - + - + + + Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome. Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 65 66 67 68 69 70 71 72 73 Male/ Female F M F F F F F F M Gestational age (weeks) 40 41 38 42 41 38 36 40 Birth weight (gram) 3150 3200 3115 2900 3310 3100 3685 1810 Intellectual disability + + + + + + + + + Developmental age at 8 months at 2+6 years at 9 months at 1+3 years at 1+3 years at 12-15 months at 13 6 months at calender age 1+4 years 6 years 2 years 11 years 7 years years 2+10 years MRI cerebrum Hypomyelination periventricular white Agnesia corpus Pachygyria and white Vermis atrophy Small brainvolume, Normal Pontocerebellar Cortical dysplasia, thin abnormalities (based matter abnormalities callosum, delayed matter abnormalities agenesia corpus Hypoplasia type 2 corpus callosum, delayed on reports) myelination callosum, abnormal myelination of right position of temporal hemisphere. lobes. Epilepsy ------+ + + Hearing impairment ------Other Factor Age at investigation 2+1 7 1 11 7 13 7 2+6 10 (years) Refraction OD S-1.75=C-0.50x26” emmetropia C-4.50x55” S+5.25=C-1.00x10” S+1.25=C-0.25x20” S+1.00 S-1.50=C-1.50x45” Refraction OS S-1.75=C-0.50x26” S+1.00=C-1.00x90” S-1.50=C-3.00x133” S+5.50=C-1.50x3” S+0.75=C-0.50x45” S+1.00 S-2.25 Correction - + + - - - - Vision 20/190 (0.10) 3/12 (0.25) LP 20/130 (0.15) 20/260 (0.08) 20/130 (0.15) 20/94 (0.2) 20/260 (0.08) 20/94 (0.2) Method TAC (55 cm) LH (3 m) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - + + + + - + - Strabismus - - + - + + + + Nystagmus + + - - - + + - - (Partial) optic disc pallor + - + - - - + + Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

124 Chromosomal aberrations in CVI

Hearing impairment ------Chapter 2.2 Other Factor Age at investigation 2+1 7 1 11 7 13 7 2+6 10 (years) Refraction OD S-1.75=C-0.50x26” emmetropia C-4.50x55” S+5.25=C-1.00x10” S+1.25=C-0.25x20” S+1.00 S-1.50=C-1.50x45” Refraction OS S-1.75=C-0.50x26” S+1.00=C-1.00x90” S-1.50=C-3.00x133” S+5.50=C-1.50x3” S+0.75=C-0.50x45” S+1.00 S-2.25 Correction - + + - - - - Vision 20/190 (0.10) 3/12 (0.25) LP 20/130 (0.15) 20/260 (0.08) 20/130 (0.15) 20/94 (0.2) 20/260 (0.08) 20/94 (0.2) Method TAC (55 cm) LH (3 m) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects - + + + + - + - Strabismus - - + - + + + + Nystagmus + + - - - + + - - (Partial) optic disc pallor + - + - - - + + Fluctuating visual + + performances G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

125 Chapter 2.2

Supplementary Table 2: Phenotype of patients with CVI and a chromosomal aberration (continued) Patient 74 75 76 77 78 79 Male/ Female M M M M F F Gestational age (weeks) 40 39 36 38 41 Birth weight (gram) 2750 2735 2475 2500 3250 Intellectual disability + + + + + + Developmental age at calender 6 months at 6 years age MRI cerebrum abnormalities Hyperdense abnormalities dorsal Dysgenesia corpus callosum and Inschaemic damage left and right (based on reports) thalamus intraventricular cyst parieto-occipital, periventricular leukomalacia, diffuse cerebral atrophy Epilepsy + + + + Hearing impairment + - Other Factor W H S S Age at investigation (years) 6 4 9 3 8 months 18 Refraction OD S+2.00 S+2.50=C-1.50x90” S-1.00=C-2.00x180” emmetropia Refraction OS S+2.00 S+2.50=C-1.50x90” S-1.50=C-2.50x180” emmetropia Correction + - - - - - Vision 20/63 (0.3) 20/130 (0.15) 20/260 (0.08) 20/1200 (0.02) 20/710 (0.03) 20/960 (0.02) Method TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects + - - + - - Strabismus + + - + + Nystagmus + + - + - + (Partial) optic disc pallor - - + - - Fluctuating visual performances + G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

126 Chromosomal aberrations in CVI

Other Factor W H S S Chapter 2.2 Age at investigation (years) 6 4 9 3 8 months 18 Refraction OD S+2.00 S+2.50=C-1.50x90” S-1.00=C-2.00x180” emmetropia Refraction OS S+2.00 S+2.50=C-1.50x90” S-1.50=C-2.50x180” emmetropia Correction + - - - - - Vision 20/63 (0.3) 20/130 (0.15) 20/260 (0.08) 20/1200 (0.02) 20/710 (0.03) 20/960 (0.02) Method TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC (55 cm) Visual field defects + - - + - - Strabismus + + - + + Nystagmus + + - + - + (Partial) optic disc pallor - - + - - Fluctuating visual performances + G=hypoglycaemia, H=hydrocephalus, LH= Lea Hyvarinen, LP =light perception, M=meningitis/encephalitis, P=perinatal problems , S=stroke, TAC= Teller Acuity Cards, W=West syndrome.

127

Chapter 3

Identifying genes for CVI

3.1

Novel genetic causes for cerebral visual impairment

Daniëlle G.M. Bosch, F. Nienke Boonstra, Nicole de Leeuw, Rolph Pfundt, Willy M. Nillesen, Joep de Ligt, Christian Gilissen, Shalini Jhangiani, James R. Lupski, Frans P.M. Cremers, Bert B. A. de Vries.

Eur J Hum Genet. Epub ahead of print. Chapter 3.1

Abstract

Cerebral visual impairment (CVI) is a major cause of low vision in children due to impairment in projection and/or interpretation of the visual input in the brain. Although acquired causes for CVI are well known, genetic causes underlying CVI are largely unidentified. DNAs of 25 patients with CVI and intellectual disability, but without acquired (e.g perinatal) damage, were investigated by whole exome sequencing. The data were analysed for de novo, autosomal recessive and X-linked variants, and subsequently, classified into known, candidate or unlikely to be associated with CVI. This classification was based on the Online Mendelian Inheritance in Man database, literature reports, variant characteristics and functional relevance of the gene. After classification, variants in four genes known to be associated with CVI (AHDC1, NGLY1, NR2F1, PGAP1) in five patients (20%) were identified, establishing a conclusive genetic diagnosis for CVI. In addition, in 11 patients (44%) with CVI, variants in one or more candidate genes were identified (ACP6, AMOT, ARHGEF10L, ATP6V1A, DCAF6, DLG4, GABRB2, GRIN1, GRIN2B, KCNQ3, KCTD19, RERE, SLC1A1, SLC25A16, SLC35A2, SOX5, UFSP2, UHMK1, ZFP30). Our findings show that diverse genetic causes underlie CVI; some of which will provide insight into the biology underlying this disease process.

132 Novel genetic causes for CVI

Introduction

Cerebral visual impairment (CVI) is one of the major causes of visual impairment in West- ern countries, as it accounts for 27% of low vision in childhood.53 CVI is a collective term of visual disorders, resulting from damage or malfunctioning of cerebral parts of the visual system, such as the optic tracts, optic radiations and the visual cortex. It is diagnosed when no ocular abnormality can explain the impairment in vision, which can consist of a reduced visual acuity, and/or visual field defects.47 In addition, abnormal visual behaviour, such as staring into light or delayed fixation can be present. Deficits in higher perceptual functions, for example difficulties with recognition of objects and faces, or visio-spatial disorders can occur, and are sometimes the only features of CVI.46,50,85 CVI can occur in isolation, but more often additional features are present, such as intellectual disability (ID), epilepsy and/or deafness.51,79,250 An important cause of CVI is acquired damage to the brain, mainly the result of perinatal problems (e.g. cerebral haemorrhage or periventricular leukomalacia), but also other types of acquired damage, such as congenital infection, hypoglycaemia, meningitis or head trauma can be causal.47 Furthermore, West syndrome and hydrocephalus can result in 115,116 CVI. So far, less attention has been paid to genetic causes of CVI, although associations Chapter 3.1 with several neurodegenerative causes and chromosomal aberrations have been described.62 Recently, we reported in 7% of CVI patients associations with copy number variants, among others trisomy 21, 1p36 deletion, and 22q13.3 deletion (Phelan-McDermid syndrome).251 Moreover, CVI was recently shown to be caused by de novo variants in NR2F1, leading to the Bosch-Boonstra-Schaaf optic atrophy syndrome (#615722, http://www.NR2F1gene.com).191 In other neurological disorders, such as ID, epileptic encephalopathies or autism, a high rate of (probably) disease causing de novo variants were identified by whole exome sequencing (WES) by using a child-parents trio approach.179,180,182-185,252 In addition, WES has also shown to be a powerful tool for identifying autosomal recessive and X-linked variants in persons with ID.170,183,184,253-255 Here, we used WES to identify underlying genetic causes for CVI.

Subjects and methods

Twenty-five patients with CVI and a visual acuity ≤0.3 were included and WES was performed in the patients and their parents (detailed methods are presented in the Supplementary Meth- ods). After performing quality filtering the common variants (>1%) were excluded, and the data were analysed for variants following a de novo, X-linked and autosomal recessive inheritance pattern. Truncating variants and variants predicted to affect function were validated by Sanger sequencing. The genes in which the variants were identified were further classified partly based on the method reported by de Ligt et al. and Gilissen et al. (Figure 1).182,256

133 Chapter 3.1

Identi ed aberrant genes (n=34) • De novo (n=22) • Autosomal recessive (n=11) • X-linked recessive (n=1)

OMIM disease gene? no (n=20)

yes (n=14)

Corresponding • Phenotype no (n=6) • Inheritance pattern • Mutation type GRIN1* ≥ 2 following items • PhyloP >3.5 / truncating† no (n=5) yes (n=7) • Enriched GO term • Enriched MP term • Brain expression Gene previously associated with CVI? no (n=3) yes (n=16)

yes (n=4)

Known CVI-associated gene (n=4) Candidate gene for CVI (n=19) Unlikely to be related to CVI (n=11) • De novo (n=2) • De novo (n=16) • De novo (n=4) • Autosomal recessive (n=2) • Autosomal recessive (n=3) • Autosomal recessive (n=6) • X-linked recessive (n=0) • X-linked recessive (n=0) • X-linked recessive (n=1)

Figure 1: Flow chart of gene classification *Inheritance does not fit with the reported OMIM disease (autosomal recessive instead of the reported de novo autosomal dominant). However, this variant is further classified according to de Ligt and Gilissen et al, see result section. †Frameshift, nonsense or splice site variant.

A second, more stringent filtering was used for thede novo and X-linked variants (frequency ≤0.1% in controls) and truncating variants (frequency of truncating variants in controls across the whole gene ≤0.1% or ≤1.0% (autosomal recessive) in controls). This study was approved by the Ethics Committee of the Radboud university medical center (Commissie Mensgebon- den Onderzoek, regio Arnhem-Nijmegen), and written informed consent was obtained for all enrolled subjects. Three patients (12, 13, and 23) were part of previous reports.191,257 The variants identified have been submitted to the Leiden Open Variation Databases (LOVDs) (http://databases.lovd.nl/, patient IDs #00025011 and #00039389 - #000394012).

Results

The clinical characteristics of the 25 patients analysed were as described in Table 1 and in more detail per patient in Supplementary Table S1. The mean age was 12 years (range 1– 33 years) and one patient had a visual acuity <0.05, which is defined as blindness by the WHO

134 Novel genetic causes for CVI

Table 1: Cohort characteristics Characteristic Number of patients Gender Male 17 Female 8 Age group <4 years 4 4-10 years 9 11-20 years 6 >20 years 6 Visual acuity ≤0.3 and ≥0.05 24 <0.05 1 Fixation abnormalities Yes 13 No 12 Visual field defects Yes 17 No 8 Developmental delay and/or intellectual disability Chapter 3.1 Yes 25 No 0 Able to walk independently Yes 15 No 10 Speak words Yes 12 No 13 Microcephaly (<3th centile) Yes 4 No 20 Unknown 1 Macrocephaly (>97th centile) Yes 3 No 21 Unknown 1 Abnormality on brain MRI Yes 14 No 8 Not assessed 3 Epilepsy Yes 9 No 16 Hearing loss Yes 1 No 23 Unknown 1

135 Chapter 3.1

(http://apps.who.int/classifications/icd10/browse/2015/en). In addition to CVI, all patients had ID, ranging from mild to severe. In all patients WES was performed by trio approach with an average read depth of 120x (Illumina Hiseq 2000, Illumina, San Diego, CA, USA), 115x (Illumina HiSeq, Illumina, San Diego, CA, USA) or 72x (Solid 5500XL, Life Technologies, Carlsbad, CA, USA). A de novo ratio could not be established, because only protein truncating variants and missense variants predicted to affect function were validated. After prioritization and validation of the identified variants in a patient and its parents, further segregation analysis for the autosomal recessive and X-linked variants was performed in the families for whom this was possible. The variants in one autosomal recessive gene, ITPRIPL1, and five variants in X-linked genes,CACNA1F, CNGA2, FLNA, PCDH11X, and ZMAT1, could be discarded because of their presence in healthy (male) family members (Supplementary Table S2). All 45 genes were classified, but 11 genes (AKAP9, ALAS2, FAM166B, KAL1, MAP3K15, MUT, POF1B, PPFIA4, SLC6A13, SPTBN5, and TRIOBP), which were excluded based on the more stringent criteria (Supplementary Table S4), are not further discussed. Of the remaining 34 genes, 14 have previously been indicated in OMIM diseases (Figure 1). For seven genes the phenotype of the patients was in line with the reported phenotype: CVI was reported previously in four genes, AHDC1, NGLY1, NR2F1, and PGAP1, whereas the other three genes were classified as candidate genes for CVI, GRIN2B, KCNQ3, and SLC35A2.191,258-260 For five genes, HSPG2, PHKB, SRP72, SYNE1 and TENM3, the reported phenotype in literature was not in line with the phenotype of the patient, and those variants were classified as unlikely to be causative for CVI in those patients (#224410, #255800, #261750, #614675, #612998, #610743, #615145). In APOPT1 the phenotype was also not in line with the reported phenotype (#220110). Moreover, we identified a de novo heterozygous variant in this gene, whereas the related OMIM disease has an autosomal recessive inheritance pattern and no second variant could be identified in the raw exome data. For another OMIM disease gene, GRIN1 (#614254), de novo heterozygous variants in this gene have been reported to cause ID; however, we identified a homozygous variant. Therefore, this variant was further classified according to the method by de Ligt et al. and Gilissen et al. as a candidate gene for CVI.182,256 This method was also used for the non-OMIM disease related genes (n=20) and consisted of the assessment of the variant characteristics (PhyloP/truncating variant) in combination with the functional relevance of the gene (GO-terms, MP-terms and brain expression), leading to a classification of 15 candidate genes for CVI and 5 genes as unlikely to be related with CVI (Figure 1). In total, four known CVI-associated genes, 19 candidate CVI genes and 11 genes unlikely to be related to CVI were identified (Table 2 and Supplementary Table S3), and in five of the 25 patients (20%) a genetic diagnosis for the CVI could be established. In another 11 patients (44%) one or more candidate genes for CVI could be identified. Photographs of the patients in whom variants in known or candidate genes were identified are shown in Figure 2 and

136 Novel genetic causes for CVI

Table 2: Identified known and candidate genes for CVI per patient Patient Dominant de novo Autosomal recessive 1 DLG4 2 NGLY1† 3 4 5 6 SLC35A2 7 AHDC1† 8 GABRB2, ARHGEF10L 9 AMOT 10 UHMK1 11 SLC25A16 GRIN1, DCAF6 12 PGAP1† 13 NR2F1† 14 15 16 17 SOX5, KCTD19 18 19 GRIN2B, ZFP30

20 ATP6V1A, UFSP2 Chapter 3.1 21 22 RERE, SLC1A1* 23 NR2F1† ACP6 24 KCNQ3 25 *Probably mosaic mutation. †Identified known CVI-associated gene.

Patient 1 Patient 2 Patient 6 Patient 7

Patient 8 Patient 9 Patient 10 Patient 17

Patient 19 Patient 20 Patient 22 Patient 24 Figure 2: Photographs of the patients in whom known or candidate genes for CVI were identified Consent to publish photographs was not obtained for patient 11.

137 Chapter 3.1 their clinical features are summarized in Table 1 and Supplementary Table S1. Pictures of patients 12, 13, and 23 have previously been published.191,257

Discussion

WES was performed in 25 patients with CVI and a visual acuity of ≤0.3, without acquired risk factors for CVI nor pathogenic copy number variants. We identified variants in four known CVI-associated genes, namely AHDC1, NGLY1, NR2F1 and PGAP1, and 19 candidate genes for CVI. In some patients multiple variants in more than one gene were found. In addition, de novo variants in NR2F1 were identified in two patients (13 and 23). The identification of variants in known CVI-associated genes strengthened our hypothesis that WES is the right approach to identify the underlying genetic causes in patients with CVI. Moreover, several identified candidate genes have a functional link with genes known to be associated with CVI. Three genes, in which variants were identified, have been implicated in glycosylation: NGLY1, SLC35A2, and PGAP1. Previously, CVI has been reported as part of congenital disorders of glycosylation (CDG) type 1a (PMM2), type 1q (SRD5A3) and type 1v (NGLY1).126,127,250,259 The phenotype of patient 2 with NGLY1 variants is similar to the previously reported patients, including the microcephaly, hypotonia, movement disorder and alacrima.259,261,262 Variants in SLC35A2 lead to CDG type 2m, featured by ID, epilepsy, facial dysmorphisms and transient abnormalities in transferin testing.263-265 In the seven reported patients with CDG type 2m, CVI has not been mentioned, but other features, including the facial dysmorphism, epilepsy and severe ID were present in patient 6. The third glycosylation gene in which a variant was identified, PGAP1 (patient 12, reported elsewhere), is important in the GPI-anchor synthesis pathway.257 Several other genes, PIGA, PIGN and PIGT, implicated in this pathway are known to be implicated in CVI and ID as well,260,266-269 and, recently, also PGAP1 variants were found to be associated with CVI and ID.260,270 RERE is another gene with a functional link with a gene known to be aberrant in CVI. A de novo variant in this arginine-glutamic acid repeats-encoding gene was identified in patient 22. RERE binds directly to NR2F1, which has recently been identified to be aberrant in Bosch- Boonstra-Schaaf optic atrophy syndrome, of which one of the features is CVI.191,271 In addition, RERE null mice show severe central nervous system abnormalities and defects of the optic vesicles.272 Furthermore, RERE forms a complex with NR2F2 and EP300 and positively regulates retinoic acid signalling in mice.273 Retinoic acid signaling induces optic vesicle and brain development and Nr2f1 and Nr2f2 transcription in mice stem cells.274 These findings indicate that RERE is a likely candidate gene for CVI (http://www.REREgene.com). Several other candidate genes, GRIN2B, GRIN1, KCNQ3, GABRB2 and SOX5, have been implicated in neurological diseases other than CVI. In GRIN2B a de novo missense variant was

138 Novel genetic causes for CVI identified in patient 19. Variants in GRIN2B have previously been found in individuals with ID.275 GRIN2B encodes the subunit NR2B of the NMDA receptor, which is present during development. In the first decade, during the critical period of developing cerebral visual cortex, NR2B is replaced by NR2A, encoded by GRIN2A.276 For GRIN2A one 4-year-old girl has been reported with low vision.277 So it might be expected that variants in GRIN2B can lead to a disturbed development and subsequently CVI. So far, 18 patients with GRIN2B variants have been reported.180,181,252,275,278-280 In two patients with West syndrome poor eye contact was reported,279 whereas in the other patients no assessment for CVI or visual acuity measurement was mentioned. In patient 11 a homozygous missense variant in GRIN1 was identified. GRIN1 encodes for NR1, which, together with NR2B or NR2A, forms the NMDA-receptor. NR1 is an essential subunit for the NMDA-receptor, and full Nr1 knockout mice are not viable.281 So far, only four patients with de novo heterozygous variants in GRIN1 have been reported with ID with or without epilepsy.180,282,283 Those variants are located in or nearby the transmembrane domains,282 in contrast to the homozygous variant identified in patient 11, which is situated in the extracellular ligand-binding domain of GRIN1 (http://www.ebi.ac.uk/interpro).284 Whether the here identified variant might be considered as a hypomorphic variant, giving rise to an Chapter 3.1 autosomal recessive disorder, awaits functional proof. In patient 24, with ID and absence seizures, a de novo missense variant in KNCQ3 was identified. The same variant was previously reported in a patient with severe ID and multifocal abnormalities on EEG.185 KNCQ3 encodes a potassium channel subunit, and has been implicated in benign epileptic seizures. Several families have been reported with seizures that resolve before the age of 6 years without CVI or ID.285-293 However, recently, two additional families with seizures and variants in KCNQ3 have been reported in which family members had various IQ levels from severe ID to normal.294,295 Therefore, the phenotypic spectrum of KCNQ3 variants appeared to be broader than benign epilepsy only and might well include CVI. A de novo GABRB2 variant in the transmembrane domain was identified in patient 8. In addition to CVI and ID, by EEG he had continuous spike and wave during slow wave sleep (CSWS) epilepsy, a severe epileptic encephalopathy, from the age of 6 years. One de novo GABRB2 variant, in the N-terminal extracellular domain implicated in GABA-binding, has been reported in a patient with ID and febrile seizures, tonic clonic convulsions and partial seizures.296 Variants in other genes encoding GABA type A-receptor subunits have been identified in different epilepsy syndromes, makingGABRB2 a likely explanation for the phenotype in our patient. Finally, in patient 17 a de novo missense variant in SOX5 was identified, located in the HMG- domain, which is important for DNA- and protein binding. Intragenic deletions in SOX5 have been reported as a cause for ID.297-299 In one patient optic nerve hypoplasia was reported, which is in agreement with the slightly pale optic discs in our patient. However, several intragenic deletions are reported in healthy individuals in the Database of Genomic Variants

139 Chapter 3.1

(http://dgv.tcag.ca/dgv/app/home).147 Without functional assays it is difficult to ascertain whether the here identified missense variant leads to loss or gain of function. In literature several genes, for example PAX6 and SOX2, are reported to influence the structural development of eye and brain.300 These genes may affect other parts of the visual system as well. However, whether aberrant genes lead to a vision disorder exclusively owing to structural eye defects, or whether an additional cerebral component is present, is difficult to distinguish. In the here presented patients without structural eye abnormalities no rare variants have been identified in these genes. In total, in 5 out of the 25 patients (20%) a genetic diagnosis for the CVI could be established. The proportion of genetically solved cases is lower than previously reported for ID, but this is probably due to the fact that only a few CVI genes are yet known.182-185 In another 11 patients (44%) variants in one or more candidate genes for CVI could be identified with several showing a functional link with known genes for CVI. In addition, several candidate genes have been implicated in a neurological disorder, such as ID and epilepsy. In those reported patients ophthalmological investigation has not always been performed or mentioned, and especially in patients with ID CVI can easily remain unnoticed.225 In addition, for some disorders only few patients have been reported, and the full clinical spectrum and its variability is probably not yet clear. Nevertheless, for the identified candidate genes reported here it is of importance to find more patients with CVI and a variant in the same gene to establish a causal relationship. In combination with additional functional studies this will increase our insight into the development of the visual system. So far, CVI has been mainly investigated in the light of acquired brain damage. Previously, we associated several chromosomal aberrations and NR2F1 with CVI.191,251 Here, we show the importance of monogenetic disorders in the pathogenesis of CVI and the necessity to test for genetic defects using genome-wide diagnostic tools.

140 Novel genetic causes for CVI

Supplementary methods

Subjects Twenty-five patients with CVI and a visual acuity ≤0.3 were included. The history was taken, and no potential risk factors for CVI, such as preterm birth, perinatal problems or hydrocephalus, were present. CVI was diagnosed when no other ocular diagnosis could explain the visual impairment. The diagnosis was made by a paediatric ophthalmologist after ophthalmological examination. This examination included visual acuity testing, crowding measurements, the examination of eye movements, fixation and oculomotor abnormalities, visual field measurements, slit lamp examination, and funduscopy. In patient with a lower cognitive level visual acuity was measured with forced preferential looking by using Teller acuity cards (TAC).64 In patients with higher developmental level tests based on object recognition, such as the LH test or Landolt C test were used.66 Visual fields were measured by using a confrontational method with white Stycar balls on a stick.75 In addition, the patients were clinically examined by a clinical geneticist and pathogenic chromosomal aberrations were excluded by array CGH. WES was performed in the patients and their parents, and except for patient 11, the parents were unrelated. Chapter 3.1 Whole exome sequencing In all 25 patients WES was performed using the trio approach (patient-parents).179 In 11 trios (patients 1-11) WES was performed on an Illumina HiSeq platform with the Agilent SureSelect All Exon V4 reagent for target enrichment (Agilent Technologies, Inc., Santa Clara, CA, USA). Alignment and variants and indels were called with Burrows-Wheeler Aligner, BWA, and Genome Analysis Toolkit, GATK.301,302 WES was executed in 10 trios (patients 12-21) at the Baylor-Hopkins Center for Mendelian Genomics on a Illumina Hiseq 2000 platform (Illumina, San Diego, CA, USA) and in three trios (patients 22, 23 and 24) on a Solid 5500 XL platform (Life Technologies, Carlsbad, CA, USA) of which the methods have been reported previously.191,303 In one trio (patient 25) the WES was also performed on a Solid 5500 XL platform, but the Agilent XL SureSelect All Exon 50Mb reagent was used for target enrichment (Agilent Technologies, Inc., Santa Clara, CA, USA) and the variants and indels were called with Lifescope v2.1 (Life Technologies, Carlsbad, CA, USA). In all 25 patients and their parents the sequence reads were mapped and aligned to the USCS genome Browser GRCh37/hg19 Human Genome Reference Assembly.

Variant prioritization and validation After quality filtering (variant reads >15%) local and global variants (≥1% allele occurrence in intramural database, dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) or Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (http://evs.gs.washington.edu/EVS/) were excluded (Supplementary Figure S1). For the 10 patients for which WES was performed at the Baylor-Hopkins Center for Mendelian Genomics, the data were also filtered for cohort allele frequency ≤2% (cohort consisted of patients with various disorders and their healthy parents, n=200) and an additional quality score was used (PHRED ≥80 or passed). The exonic nonsyn-

141 Chapter 3.1 onymous and canonical splice site variants were selected for further analysis. A de novo analysis was performed for all trios. Furthermore, the results were analysed for homozygous variants (>80% variant reads), compound heterozygous (two or more variants present in one gene) and hemizygous variants in males (X-chromosome, >80% variant reads). When the autosomal recessive variants were present on one allele, the variants were excluded (based on the raw data (BAM-file) of a patient and its parents). Truncating variants, consisting of frameshift, nonsense or splice site variants (assessed using Interac- tive Biosoftware Alamut version 2.3 rev2), and missense variants predicted to affect function (majority vote of Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), MutPred (http://mutpred.mutdb.org/) and SNPs&GO (http://snps.path.uab.edu/snps-and-go/pages/method.html)) were validated by Sanger sequencing in patient and parents.304-306 For compound heterozygous variants at least one of the variants should be predicted to be protein truncating, or, when concerning missense variants, both variants should be predicted to affect function. For the maternally inherited variants on the X-chromosome in males and the autosomal recessive variants further segregation analysis in the family was undertaken, whenever possible.

Gene classification For the validated variants in genes that were previously indicated in the Online Mendelian Inheri- tance in Man (OMIM) database (www.omim.org) the inheritance pattern and the variant type were compared. Furthermore, a phenotypic comparison of the patient and the reported individuals in literature was performed by a clinical geneticist and assessment was made whether the phenotype showed similarities. If the phenotype was distinct the gene was classified as unlikely to be causative. When the phenotype showed similarities and the patients reported did not have CVI, the gene was classified as a candidate gene for CVI. Genes that were previously reported to be involved in the pathogenesis of CVI were classified as “known CVI-associated gene”. The remaining (non-OMIM disease related) validated variants were classified based on the previously reported method by De Ligt et al. and Gilissen et al.182,256 In brief, the variants were scored for their functional relevance on four items. First, it was assessed whether the variant was disruptive or whether the missense variants involved a conserved nucleotide (phyloP >3.5). Subsequently, the list of 525 genes known to be associated with ID from Gilissen et al. was loaded into ToppGene to select Gene Ontology (GO) and Mouse Phenotype (MP) terms with a significant enrichment (FDR<0.05 by Benjamini Hochberg method) (https://toppgene.cchmc.org/, January 2015).256,307 When there was overlap between the enriched terms and the GO- or MP-terms for the genes identified in the present study the variant scored positive. Finally, it was assessed whether the gene was expressed during brain development in the Human Brain Transcriptome (http://hbatlas.org/).308 A gene was considered to be expressed when the Log2 intensity was ≥6 for at least three periods of the developing brain (<20 years of age). When the detected variant and/or the gene scored positive for at least two items (conservation/disruptive, brain expression, GO- and/or MP term) the aberrant gene was classified as a possible candidate gene for CVI. Otherwise the aberrant gene was classified as unlikely to be causative for CVI.

142 Novel genetic causes for CVI

Stringent criteria For the de novo and X-linked missense variants, the variants with an allele frequency ≥0.1% in intramural database, dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) or Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (http://evs.gs.washington.edu/ EVS/) were excluded. This frequency of 0.1% is, with a wide margin, based on the estimated incidence of 0.0225% of CVI in children in the Netherlands.53 In addition, for loss of function variants, the ExAC browser (Exome Aggregation Consortium (ExAC), Cambridge, MA (http:// exac.broadinstitute.org) [(June, 2015 accessed]) was used to obtain all the loss of function variants in the identified gene. Genes with a truncating allele frequency ≥0.1% (de novo or X-linked variants) or ≥1.0% (autosomal recessive variants) were excluded.

Supplementary figures AND TABLES

Quality ltering • Variant reads >15%

• PHRED1 or 2 qualityscore* ≥80 or PASS Chapter 3.1

Gene component • Exonic nonsynonymous • Canonical splice acceptor and donor site

Excluded common variants • dbSNP allele frequency <1% • Intramural allele frequency <1% • Cohort allele frequency* ≤2%

Autosomal recessive • Homozygous (>80% variant reads) X-linked recessive in males Dominant (De novo) • Compound heterozygous (>80% variant reads) (bi-allelic mutations)

Missense variant: Truncating variant† majority vote: aect function (Mutpred, SNPs&Go, PolyPhen-2)

Validation & segregation analysis

Supplementary Figure S1: Flow chart of variant prioritization *Only used for the 10 patients in whom WES was performed at the Baylor-Hopkins Center for Mendelian Genomics. †Frameshift, nonsense or splice site variant.

143 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed. Patient 1 2 3 4 Gender M M M M Birth weight (grams) 3640 (75th centile) 2415 (15th centile) Normal 3750 Gestational age (weeks) 39 37 41 At term Age at investigations (years) 5 3+7 33 30 Height (cm) 118 cm (50th centile) 97 (15th centile) OFC (cm) 51.5 cm (40th centile) 47.0 (1st centile) 55.5 (10th centile) 56.0 (15th centile) Development 2-3 years at 6 years 11 months at 1+9 months Delayed Delayed First words 9 months - - - Walk independently 1+10 years - 10 years - Dysmorphisms Long eyelashes, full nasal tip, long columella, thin Metopic ridge, medial flaring of Coarse facial features, prominente ridge above the eyes, upward Deepset eyes, prominent nose, small nostrils, full alae nasi, upper lip, large ears, fragmented palmar creases, eyebrows, long upward slanted slanted palpebral fissures, broad nasal ridge, protruding ears, broad chin, single tranverse palmar crease right, tapering short distal finger phalanges, longitudinally grooved palpebral fissures, small mouth, extension restriction of knees and fingers, hypoplastic toenails fingers, broad proximal interphalangeal joints, long toes and fingernails, fetal finger pads, short distal toe small ears, tapering fingers, overriding II toes phalanges, syndactyly II-III toes, clinodactyly IV-V toes bilateral clinodactyly IV and V toes, hypoplastic toenails, extension restriction of knee, small penis Hearing Normal Normal Normal Normal Other abnormalities Autism, hypermobility Alacrima, cryptorchidism, feeding - Automutilation, recurrent otitis problems, hypotonia, involuntary movements, axonal polyneuropathy Epilepsy - - + + Report MRI brain Normal Delayed myelination NP NP Refraction error, right eye S+0.25 S+1.00 S+1.00=C-1.00x90” S-3.00=C-1.00x45” Refraction error, left eye S+0.50 Emmetropic S+1.00=C-1.00x90” S-3.00=C-1.00x135” Correction of refraction - - - - Visual acuity, both eyes 20/63 (0.3) 20/190 (0.1) 20/260 (0.08) 0.1 Method (distance) TAC (55 cm) TAC (55 cm) TAC (55 cm) Cardiff (1 m) Strabismus - + + + Motility disorder of the eyes not - + + + leading to strabismus Nystagmus Manifest - - - Visual field defects - + - + Slit lamp examination Iris transillumination Cornea scar Normal Normal Optic disc Partial pale Partial pale Normal Pale Macula Normal NP Normal Normal VEP (age) NP NP Flash: prolonged latency time. Pattern: normal (10 years) NP ERG (age) NP NP NP Visual behaviour, fixation and visual Fluctuating visual attention, looking away from the target, short Short fixation attention fixation, auditive stimuli dominates

144 Novel genetic causes for CVI

Hearing Normal Normal Normal Normal Chapter 3.1 Other abnormalities Autism, hypermobility Alacrima, cryptorchidism, feeding - Automutilation, recurrent otitis problems, hypotonia, involuntary movements, axonal polyneuropathy Epilepsy - - + + Report MRI brain Normal Delayed myelination NP NP Refraction error, right eye S+0.25 S+1.00 S+1.00=C-1.00x90” S-3.00=C-1.00x45” Refraction error, left eye S+0.50 Emmetropic S+1.00=C-1.00x90” S-3.00=C-1.00x135” Correction of refraction - - - - Visual acuity, both eyes 20/63 (0.3) 20/190 (0.1) 20/260 (0.08) 0.1 Method (distance) TAC (55 cm) TAC (55 cm) TAC (55 cm) Cardiff (1 m) Strabismus - + + + Motility disorder of the eyes not - + + + leading to strabismus Nystagmus Manifest - - - Visual field defects - + - + Slit lamp examination Iris transillumination Cornea scar Normal Normal Optic disc Partial pale Partial pale Normal Pale Macula Normal NP Normal Normal VEP (age) NP NP Flash: prolonged latency time. Pattern: normal (10 years) NP ERG (age) NP NP NP Visual behaviour, fixation and visual Fluctuating visual attention, looking away from the target, short Short fixation attention fixation, auditive stimuli dominates

145 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 5 6 7 8 Gender M F F M Birth weight (grams) 4250 (95th centile) 3560 (70th centile) Gestational age (weeks) 39 39 43 40 Age at investigations (years) 27 23 22 17 Height (cm) 190 (84th centile) 167 (30th centile) OFC (cm) 59.0 (70th centile) 50.5 (<1st centile) 59.0 (99th centile) Development PIQ = 55-69 and VIQ = 35-40 Delayed Delayed Delayed First words 2-2+6 years - 4 years - Walk independently 2-2+6 years - + 7 years Dysmorphisms Synophrys, full eyebrows, high palate, retrognathia, Full eyebrows, stiff hair, mid facial Long asymmetric face, proptosis, broad nose, full lips, broad Broad nose, full lips, large hands, broad fingers prominent ear lobes, broad distal phalanges, broad hypoplasia, full lips, soft skin of the mouth, wide space teeth, small high palate, simple left ear, hallux hands, hyperlaxity of the fingers overfolding of the right superior helices, hyperlordosis, soft skin, IV finger clinodactyly, fetal finger pads, pes equines, many moles Hearing Normal Normal Normal Normal Other abnormalities Recurrent otitis, PDD-NOS, ADHD, hypotonia Unilateral congenital talipes Hypotonia, adenotonsillectomy Gastroesophageal reflux, autism equinovarus, hypotonia, scoliosis, swallowing problems resulting into PEG tube for fluid Epilepsy + + + + Report MRI brain Normal Enlarged peripheral liquor system, Asymmetric wide ventricles, thin corpus callosum NP delayed myelination Refraction error, right eye S+2.00=C-1.00x180” S+1.00=C-2.50x153” S+0.50=C-1.00x66” S-6.00 Refraction error, left eye S+1.75=C-1.25x180” S-1.00=C-3.50x12” S+1.00=C-1.75x91” S-6.00 Correction of refraction - - - - Visual acuity, both eyes 3/10 (0.3) 20/190 (0.1) 3/12 (0.25) 20/260 (0.06) Method (distance) Landolt C (5 m) TAC (55 cm) LH (3 m) TAC (55 cm) Strabismus - - + + Motility disorder of the eyes not + + + - leading to strabismus Nystagmus Manifest and latent - Manifest and latent - Visual field defects + + - + Slit lamp examination Normal Normal Normal Normal Optic disc Partial pale Partial pale Normal Pale, enlarged excavations Macula Not recognizable Normal Normal Normal VEP (age) Flash: normal. Pattern: normal (11 years) Pattern: normal. Flash: variable NP NP response with normal latency time (1+8 years) ERG (age) Normal (13 years) NP Normal (2 months) NP Visual behaviour, fixation and visual Fixation problems, looking away from the target, Fixation abnormalities Does not use his vision attention periods of diminished visual attention

146 Novel genetic causes for CVI

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 5 6 7 8 Gender M F F M Birth weight (grams) 4250 (95th centile) 3560 (70th centile) Gestational age (weeks) 39 39 43 40 Age at investigations (years) 27 23 22 17 Height (cm) 190 (84th centile) 167 (30th centile) OFC (cm) 59.0 (70th centile) 50.5 (<1st centile) 59.0 (99th centile) Development PIQ = 55-69 and VIQ = 35-40 Delayed Delayed Delayed First words 2-2+6 years - 4 years - Walk independently 2-2+6 years - + 7 years Dysmorphisms Synophrys, full eyebrows, high palate, retrognathia, Full eyebrows, stiff hair, mid facial Long asymmetric face, proptosis, broad nose, full lips, broad Broad nose, full lips, large hands, broad fingers prominent ear lobes, broad distal phalanges, broad hypoplasia, full lips, soft skin of the mouth, wide space teeth, small high palate, simple left ear, hallux hands, hyperlaxity of the fingers overfolding of the right superior helices, hyperlordosis, soft skin, IV finger clinodactyly, fetal finger pads, pes equines, many moles Hearing Normal Normal Normal Normal Other abnormalities Recurrent otitis, PDD-NOS, ADHD, hypotonia Unilateral congenital talipes Hypotonia, adenotonsillectomy Gastroesophageal reflux, autism equinovarus, hypotonia, scoliosis, Chapter 3.1 swallowing problems resulting into PEG tube for fluid Epilepsy + + + + Report MRI brain Normal Enlarged peripheral liquor system, Asymmetric wide ventricles, thin corpus callosum NP delayed myelination Refraction error, right eye S+2.00=C-1.00x180” S+1.00=C-2.50x153” S+0.50=C-1.00x66” S-6.00 Refraction error, left eye S+1.75=C-1.25x180” S-1.00=C-3.50x12” S+1.00=C-1.75x91” S-6.00 Correction of refraction - - - - Visual acuity, both eyes 3/10 (0.3) 20/190 (0.1) 3/12 (0.25) 20/260 (0.06) Method (distance) Landolt C (5 m) TAC (55 cm) LH (3 m) TAC (55 cm) Strabismus - - + + Motility disorder of the eyes not + + + - leading to strabismus Nystagmus Manifest and latent - Manifest and latent - Visual field defects + + - + Slit lamp examination Normal Normal Normal Normal Optic disc Partial pale Partial pale Normal Pale, enlarged excavations Macula Not recognizable Normal Normal Normal VEP (age) Flash: normal. Pattern: normal (11 years) Pattern: normal. Flash: variable NP NP response with normal latency time (1+8 years) ERG (age) Normal (13 years) NP Normal (2 months) NP Visual behaviour, fixation and visual Fixation problems, looking away from the target, Fixation abnormalities Does not use his vision attention periods of diminished visual attention

147 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 9 10 11 12 Gender F M F M Birth weight (grams) 3300 (40th centile) 4250 (85th centile) 2900 3550 gram (70th centile) Gestational age (weeks) 40 42 At term 38 Age at investigations (years) 13 12 6 7 Height (cm) 161 (50th centile) 131 (50th centile) OFC (cm) 54.0 (50th centile) 55.0 (85th centile) 50.5 (35th centile) 52.5 (50th centile) Development 1+6-2+6 years at 5+7 years < 24 months Delayed IQ 49 First words 2 years - - 2-2+6 years Walk independently 2+6 years - - 2-2+6 years Dysmorphisms Large, slightly protruding ears, broad nasal tip with Upward slanted palpebral fissures, Upward slanted palpebral fissures, full nasal tip and alae nasi, Upward slanted palpebral fissures, deep set eyes, large ear lobes, vertical dimple in nasal ridge, high palate, full lips, deep-set eyes, broad nose, large large tongue, broad alveolar ridges prominent helices and antihelices, tooth had extra mammelons, long fingers, additional flexion creases of III fingers, left ear, prominent crux superior, diminished enamel fragmented palmar creases, long hallux, long small fetal finger pads, pes equines, hallux feet, dry skin, longtitudinal creases in fingernails valgus, syndactyly II-III toe Hearing Normal Mild increased hearing treshold Normal Normal Other abnormalities Autism Bilateral cheilognathopalatoschisis, Central hypotonia, recurrent upper airway infections, feeding Hypotonia, factor XII deficiency gastroesophageal reflux, periods of problems restlessness and sleeping problems Epilepsy - - - - Report MRI brain Cerebellar atrophy and to a less extent of cerebrum Cavum septum pellucidum, thin Probably white matter abnormalities (poor quality scan due Normal corpus callosum to movement) Refraction error, right eye S+5.25=C-1.00x95” S+2.00=C-0.75x8” S+3.25=C-3.75x20” Emmetropic Refraction error, left eye S+3.00=C-0.25x90” S+1.50=C-1.00x3” S+4.25=C-4.25x166” Emmetropic Correction of refraction + - - - Visual acuity, both eyes 10/32 (0.3) 20/130 (0.15) 20/190 (0.1) 3/10 (0.3) Method (distance) LH (3 m) TAC (55 cm) TAC (55 cm) Landolt C (5 m) Strabismus + + + + Motility disorder of the eyes not + - + leading to strabismus Nystagmus - - - Latent, and minimal manifest Visual field defects - + + - Slit lamp examination Normal Normal Megalocornea Normal Optic disc Normal Normal Normal Normal Macula Normal Normal Normal Normal VEP (age) NP NP Flash: normal. Pattern reversal: normal (10 months) NP ERG (age) NP NP Normal (9 months) NP Visual behaviour, fixation and visual Fixation abnormalities Fluctuating visual attention, slow Staring at lights Crowding, short fixation attention visual reaction

148 Novel genetic causes for CVI

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 9 10 11 12 Gender F M F M Birth weight (grams) 3300 (40th centile) 4250 (85th centile) 2900 3550 gram (70th centile) Gestational age (weeks) 40 42 At term 38 Age at investigations (years) 13 12 6 7 Height (cm) 161 (50th centile) 131 (50th centile) OFC (cm) 54.0 (50th centile) 55.0 (85th centile) 50.5 (35th centile) 52.5 (50th centile) Development 1+6-2+6 years at 5+7 years < 24 months Delayed IQ 49 First words 2 years - - 2-2+6 years Walk independently 2+6 years - - 2-2+6 years Dysmorphisms Large, slightly protruding ears, broad nasal tip with Upward slanted palpebral fissures, Upward slanted palpebral fissures, full nasal tip and alae nasi, Upward slanted palpebral fissures, deep set eyes, large ear lobes, vertical dimple in nasal ridge, high palate, full lips, deep-set eyes, broad nose, large large tongue, broad alveolar ridges prominent helices and antihelices, tooth had extra mammelons, long fingers, additional flexion creases of III fingers, left ear, prominent crux superior, diminished enamel fragmented palmar creases, long hallux, long small fetal finger pads, pes equines, hallux feet, dry skin, longtitudinal creases in fingernails valgus, syndactyly II-III toe Hearing Normal Mild increased hearing treshold Normal Normal Other abnormalities Autism Bilateral cheilognathopalatoschisis, Central hypotonia, recurrent upper airway infections, feeding Hypotonia, factor XII deficiency gastroesophageal reflux, periods of problems Chapter 3.1 restlessness and sleeping problems Epilepsy - - - - Report MRI brain Cerebellar atrophy and to a less extent of cerebrum Cavum septum pellucidum, thin Probably white matter abnormalities (poor quality scan due Normal corpus callosum to movement) Refraction error, right eye S+5.25=C-1.00x95” S+2.00=C-0.75x8” S+3.25=C-3.75x20” Emmetropic Refraction error, left eye S+3.00=C-0.25x90” S+1.50=C-1.00x3” S+4.25=C-4.25x166” Emmetropic Correction of refraction + - - - Visual acuity, both eyes 10/32 (0.3) 20/130 (0.15) 20/190 (0.1) 3/10 (0.3) Method (distance) LH (3 m) TAC (55 cm) TAC (55 cm) Landolt C (5 m) Strabismus + + + + Motility disorder of the eyes not + - + leading to strabismus Nystagmus - - - Latent, and minimal manifest Visual field defects - + + - Slit lamp examination Normal Normal Megalocornea Normal Optic disc Normal Normal Normal Normal Macula Normal Normal Normal Normal VEP (age) NP NP Flash: normal. Pattern reversal: normal (10 months) NP ERG (age) NP NP Normal (9 months) NP Visual behaviour, fixation and visual Fixation abnormalities Fluctuating visual attention, slow Staring at lights Crowding, short fixation attention visual reaction

149 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 13 14 15 16 Gender M M M M Birth weight (grams) 3510 (7th centile) 3980 (70th centile) 3520 2900 (<2th centile) Gestational age (weeks) 42 42 At term 42 Age at investigations (years) 12 3+8 1+7 21 Height (cm) 159 (p84) 162 (<1st centile) OFC (cm) 57.0 (98th centile) 53.3 (87th centile) 45.2 (1st centile) 54.5 (2nd centile) Development IQ 48 7 months at 2+7 years 5-7 months at 2+8 years Delayed First words 1 year - - - Walk independently 2 years - - 7 years Dysmorphisms Protruding ears, long nostrils, prominent alae nasi, Epicanthal folds, blepharophimosis, Broad forehead, hypertelorism, broad mouth with thin upper Upward slanting palpebral fissures, hypertelorism, prominent flattened thorax, long fingers and toes. upward slanted palpebral fissures, lip, pointed helices, prominent crus of the ear, flattened upper nose, broad mouth with full lips, small ears, protruding ear lobes, flattened nasal tip, long philtrum, helices, short broad hands, tapering fingers, fragmented flattened folded helices, prominent antihelices, soft skin of the small teeth, retrognathia, deep palmar creases hands and feet, fetal finger pads, unilateral tranverse palmar palmar creases, long tapering crease, pes equinis, deep set toenails fingers, longtitudinal creases in fingernails, long hallux Hearing Normal Normal Normal Normal Other abnormalities - Hypotonia, feeding problems, Feeding problems Hypotonia, restless, temper tantrums hyperlaxity of joints Epilepsy - - - - Report MRI brain Normal Cerebellar dysplasia and white Normal Normal matter abnormalities Refraction error, right eye S+7.25=C-2.25x164” S+0.50 S+1.50=C-1.00x180” S+1.00 Refraction error, left eye S+6.75=C-1.75x30” S+1.25=C-0.50x180” S+2.00=C-1.25x180” S-0.50 Correction of refraction + - - - Visual acuity, both eyes 10/80 (0.125) 20/260 (0.08) 20/170 (0.12) 20/1600 (0.01) Method (distance) LH (3 m) TAC (55 cm) TAC (55 cm) TAC 55 cm Strabismus + + - + Motility disorder of the eyes not - - - + leading to strabismus Nystagmus Latent Latent Manifest - Visual field defects + - + + Slit lamp examination Iris transillumination Cloudy lens Normal Normal Optic disc Small, enlarged excavation Normal Slightly small Temporal pale, enlarged excavation Macula Not recognizable Normal Normal Normal VEP (age) Flash: late response Pattern onset: late response. NP Pattern reversal: no reproducible responses NP Pattern reversal: normal respons (8 years) ERG (age) Normal (8 years) NP NP NP Visual behaviour, fixation and visual Crowding, fluctuating visual performance, excentric Staring into light attention fixation

150 Novel genetic causes for CVI

Hearing Normal Normal Normal Normal Chapter 3.1 Other abnormalities - Hypotonia, feeding problems, Feeding problems Hypotonia, restless, temper tantrums hyperlaxity of joints Epilepsy - - - - Report MRI brain Normal Cerebellar dysplasia and white Normal Normal matter abnormalities Refraction error, right eye S+7.25=C-2.25x164” S+0.50 S+1.50=C-1.00x180” S+1.00 Refraction error, left eye S+6.75=C-1.75x30” S+1.25=C-0.50x180” S+2.00=C-1.25x180” S-0.50 Correction of refraction + - - - Visual acuity, both eyes 10/80 (0.125) 20/260 (0.08) 20/170 (0.12) 20/1600 (0.01) Method (distance) LH (3 m) TAC (55 cm) TAC (55 cm) TAC 55 cm Strabismus + + - + Motility disorder of the eyes not - - - + leading to strabismus Nystagmus Latent Latent Manifest - Visual field defects + - + + Slit lamp examination Iris transillumination Cloudy lens Normal Normal Optic disc Small, enlarged excavation Normal Slightly small Temporal pale, enlarged excavation Macula Not recognizable Normal Normal Normal VEP (age) Flash: late response Pattern onset: late response. NP Pattern reversal: no reproducible responses NP Pattern reversal: normal respons (8 years) ERG (age) Normal (8 years) NP NP NP Visual behaviour, fixation and visual Crowding, fluctuating visual performance, excentric Staring into light attention fixation

151 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 17 18 19 20 Gender M F M M Birth weight (grams) 2545 (20th centile) 2910 (4th centile) 3870 (75th centile) 2820 (15th centile) Gestational age (weeks) 37 42 40 38 Age at investigations (years) 12 11 8 8 Height (cm) 162 (84th centile) 124 (10th centile) OFC (cm) 53.5 (35th centile) 57.5 (>99th centile) 51.5 (30th centile) 49.5 (5th centile) Development 5 years at 11 years Delayed Delayed 9-11 months at 2+11 years First words 3 years - - + Walk independently 2+9 5+6 - 6 years Dysmorphisms Marfanoid habitus, straight nose, full lips, Full hair, prominent forehead, Straight low set eyebrows, long eye lashes, down slanted Fetal finger pads, flat nails. fragmentated palmar creases, long fingers, fetal low-set eyebrows, epicanthal folds, palpebral fissures, large ears, cupid bow upper lip, thin upper V-shaped pads broad nasal root, short philtrum, lip, telangiectasias on back full lips, small ears, prominent antihelices, flattened helices, supernumerary nipples, fragmented palmar creases, broad proximal interphalangeal joints, long hands Hearing Normal Normal Normal NA Other abnormalities Small mandible Palatoschisis, clenched hands, Adenotonsillectomy, recurrent otitis, bilateral cryptorchidism, Amelogenesis imperfecta, otitis hypotonia, sleeping problems, hypotonia, constipation, hand biting, eye poking recurrent otitis, stereotypic movements Epilepsy - - - + Report MRI brain Normal Bifrontal pachygyria, wide virchow Normal Small areas of gliosis occipital robin spaces, dysplastic cerebellum Refraction error, right eye S+1.50=C-0.50x146” Splan=C-6.00x55” S+6.00=C-5.00x12” S+1.00 Refraction error, left eye S+1.00=C-0.25x116” S-2.50=C-2.50x135” S+4.00=C-4.00x167” S+1.25 Correction of refraction - + + - Visual acuity, both eyes 10/32 (0.3) 20/130 (0.15) 20/130 (0.15) 20/130 (0.15) Method (distance) LH (3 m) TAC (84 cm) TAC (55 cm) TAC (55cm) Strabismus + + + - Motility disorder of the eyes not + - + + leading to strabismus Nystagmus - - Manifest - Visual field defects + + + + Slit lamp examination Normal Normal Normal Normal Optic disc Slightly pale Normal Enlarged excavations Pale Macula Hypoplastic Normal Normal No clear macula reflex VEP (age) NP NP NP NP ERG (age) Normal (8 years) NP NP NP Visual behaviour, fixation and visual Crowding Fixation abnormalities Looking away from the target, fixation abnormalities attention

152 Novel genetic causes for CVI

interphalangeal joints, long hands Chapter 3.1 Hearing Normal Normal Normal NA Other abnormalities Small mandible Palatoschisis, clenched hands, Adenotonsillectomy, recurrent otitis, bilateral cryptorchidism, Amelogenesis imperfecta, otitis hypotonia, sleeping problems, hypotonia, constipation, hand biting, eye poking recurrent otitis, stereotypic movements Epilepsy - - - + Report MRI brain Normal Bifrontal pachygyria, wide virchow Normal Small areas of gliosis occipital robin spaces, dysplastic cerebellum Refraction error, right eye S+1.50=C-0.50x146” Splan=C-6.00x55” S+6.00=C-5.00x12” S+1.00 Refraction error, left eye S+1.00=C-0.25x116” S-2.50=C-2.50x135” S+4.00=C-4.00x167” S+1.25 Correction of refraction - + + - Visual acuity, both eyes 10/32 (0.3) 20/130 (0.15) 20/130 (0.15) 20/130 (0.15) Method (distance) LH (3 m) TAC (84 cm) TAC (55 cm) TAC (55cm) Strabismus + + + - Motility disorder of the eyes not + - + + leading to strabismus Nystagmus - - Manifest - Visual field defects + + + + Slit lamp examination Normal Normal Normal Normal Optic disc Slightly pale Normal Enlarged excavations Pale Macula Hypoplastic Normal Normal No clear macula reflex VEP (age) NP NP NP NP ERG (age) Normal (8 years) NP NP NP Visual behaviour, fixation and visual Crowding Fixation abnormalities Looking away from the target, fixation abnormalities attention

153 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 21 22 23 24 Gender F M F F Birth weight (grams) 3310 (20th centile) 4190 (87th centile) 3855 (70th centile) 2840 (8th centile) Gestational age (weeks) 41 40 41 40 Age at investigations (years) 7 10 2+4 4 Height (cm) 157 (98th centile) 87 (16th centile) 112 (86th centile) OFC (cm) 50.5 (16th centile) 55.5 (86th centile) 49.0 (55th centile) 49.5 (40th centile) Development Delayed 6-24 months at 9 years Delayed 11-15 months at 3+6 years First words 2+2 years - 1+6 years 3+6 years Walk independently - + - 1+9 years Dysmorphisms Ptosis, broad mouth, full lips, low hairline, small V Prominent forehead, deep-set eyes, Epicanthal folds, upturned nasal tip, broad mouth, full lips, Upward slanted palpebral fissures, blepharophimosis, pear fingers bilateral blepharophimosis, full simple protruding ears with upturned ear lobes, tapering shaped nose, full nasal tip, full everted lower lip, small teeth, long nose, full lips, broad large incisors, fingers and fetal fingers pads, curly slow growing hair tapering fingers, fetal finger pads. Straight blond hair with an broad alveolar ridges, small high occipital hair spot with dark curly hair palate, thick helices and flattened upper helices Hearing Normal Normal Normal Normal Other abnormalities Hypotonia, feeding problems, gastroesophageal Hypotonia, feeding problems, Hypotonia Recurrent upper airway infections reflux, cerebellar ataxia pyloric hypertrophy, gastroesophageal reflux, vesicourethral reflux, recurrent airway infections Epilepsy - + - + Report MRI brain Periventricular white matter abnormalities, vermis Wide ventricles, small archnoid cyst, Small optic chiasm and optic nerve, white matter abnormalities Bilateral subcortical periventricular white matter abnormalities atrophy possible frontal falx agenesis periventricular parieto-occipitaal Refraction error, right eye S+5.25=x-1.00x10” Emmetropic S+3.00 S+1.00 Refraction error, left eye S+5.50=C-1.50x3” Emmetropic S+3.00 S+1.00 Correction of refraction + - - - Visual acuity, both eyes 20/260 (0.08) 20/380 (0.05) 20/260 (0.08) 20/260 (0.08) Method (distance) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC 955cm) Strabismus - + + + Motility disorder of the eyes not + - - + leading to strabismus Nystagmus - - - - Visual field defects + - + + Slit lamp examination No pupilreflex, mild lens clouding Normal Normal Normal Optic disc Normal Pale Pale Partial pale Macula Normal Immature reflex Normal Normal VEP (age) NP NP Flash: long latency times (10 months) NP ERG (age) NP NP NP NP Visual behaviour, fixation and visual Fixation abnormalities Reaching at target without looking Short fixation, fluctuating visual attention attention

154 Novel genetic causes for CVI

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 21 22 23 24 Gender F M F F Birth weight (grams) 3310 (20th centile) 4190 (87th centile) 3855 (70th centile) 2840 (8th centile) Gestational age (weeks) 41 40 41 40 Age at investigations (years) 7 10 2+4 4 Height (cm) 157 (98th centile) 87 (16th centile) 112 (86th centile) OFC (cm) 50.5 (16th centile) 55.5 (86th centile) 49.0 (55th centile) 49.5 (40th centile) Development Delayed 6-24 months at 9 years Delayed 11-15 months at 3+6 years First words 2+2 years - 1+6 years 3+6 years Walk independently - + - 1+9 years Dysmorphisms Ptosis, broad mouth, full lips, low hairline, small V Prominent forehead, deep-set eyes, Epicanthal folds, upturned nasal tip, broad mouth, full lips, Upward slanted palpebral fissures, blepharophimosis, pear fingers bilateral blepharophimosis, full simple protruding ears with upturned ear lobes, tapering shaped nose, full nasal tip, full everted lower lip, small teeth, long nose, full lips, broad large incisors, fingers and fetal fingers pads, curly slow growing hair tapering fingers, fetal finger pads. Straight blond hair with an broad alveolar ridges, small high occipital hair spot with dark curly hair palate, thick helices and flattened upper helices Hearing Normal Normal Normal Normal Other abnormalities Hypotonia, feeding problems, gastroesophageal Hypotonia, feeding problems, Hypotonia Recurrent upper airway infections Chapter 3.1 reflux, cerebellar ataxia pyloric hypertrophy, gastroesophageal reflux, vesicourethral reflux, recurrent airway infections Epilepsy - + - + Report MRI brain Periventricular white matter abnormalities, vermis Wide ventricles, small archnoid cyst, Small optic chiasm and optic nerve, white matter abnormalities Bilateral subcortical periventricular white matter abnormalities atrophy possible frontal falx agenesis periventricular parieto-occipitaal Refraction error, right eye S+5.25=x-1.00x10” Emmetropic S+3.00 S+1.00 Refraction error, left eye S+5.50=C-1.50x3” Emmetropic S+3.00 S+1.00 Correction of refraction + - - - Visual acuity, both eyes 20/260 (0.08) 20/380 (0.05) 20/260 (0.08) 20/260 (0.08) Method (distance) TAC (55 cm) TAC (55 cm) TAC (55 cm) TAC 955cm) Strabismus - + + + Motility disorder of the eyes not + - - + leading to strabismus Nystagmus - - - - Visual field defects + - + + Slit lamp examination No pupilreflex, mild lens clouding Normal Normal Normal Optic disc Normal Pale Pale Partial pale Macula Normal Immature reflex Normal Normal VEP (age) NP NP Flash: long latency times (10 months) NP ERG (age) NP NP NP NP Visual behaviour, fixation and visual Fixation abnormalities Reaching at target without looking Short fixation, fluctuating visual attention attention

155 Chapter 3.1

Supplementary Table S1: Additional clinical characteristics of the patients in which whole exome sequencing was performed (continued) Patient 25 Gender M Birth weight (grams) 3180 (15th centile) Gestational age (weeks) 41 Age at investigations (years) 7 Height (cm) OFC (cm) 50.0 (15th centile) Development 2+6 years at 6+9 years First words + Walk independently 1+9 years Dysmorphisms Ptosis, upward slanted palpebral fissures, broad nasal rige, bifid nasal tip, full lips, prominent incisors with extra mammelons, prominent uvula, simple ears with squared helices, prominent antihelices, hypermobility of fingers and elbows, long fongers with distal brachydactyly, fragmented palmar creases, clinodactyly IV-V toes, shawl scrotum Hearing Normal Other abnormalities Polyhydramnion, hypotonia, drooling, feeding problems, bilateral hernia inguinalis, bilateral cryptorchidism, hyperactive behavior Epilepsy - Report MRI brain Periventricular white matter abnormalities Refraction error, right eye Emmetropic Refraction error, left eye S+1.00=C-1.00x90” Correction of refraction - Visual acuity, both eyes 3/12 (0.25) Method (distance) LH (3 m) Strabismus - Motility disorder of the eyes not leading to + strabismus Nystagmus - Visual field defects - Slit lamp examination Normal Optic disc Pale Macula Not seen VEP (age) Pattern: normal (6 months) ERG (age) Normal (6 months) Visual behaviour, fixation and visual attention Fixation abnormalities ADHD: attention deficit hyperactivity disorder. ERG: Electroretinography. LH: Lea Hyvärinen test eye charts. NA: Not available. NP: Not performed. OFC: Occipital frontal head circumference. PDD-NOS: Pervasive developmental disorder-not otherwise specified. PEG: Percutaneous endoscopic gastrostomy. PIQ: -Perfor mance IQ. TAC: Teller acuity cards. VEP: Visual evoked potential. VIQ: Verbal IQ.

156

Chapter 3.1

Supplementary Table S2: Not segregating variants Patient Gene Inheritance Segregation in Refseq accession OMIM database Chromosomal position DNA change Protein change dbSNP allele EVS allele Intramural allele anamnestically number reference numbers (Hg 19) frequency (%) frequency (%) frequency (%) healthy family members Present in maternal 2 FLNA X, maternal NM_001456.3 chrX:153577258 c.7879G>A p.(Glu2627Lys) 0 0.0098 0 grandfather *300017 Present in maternal 14 ZMAT1 X, maternal NM_001011657.3 chrX:101141628 c.581T>A p.(Met194Lys) 0 0 0 grandfather Present in maternal 14 CNGA2 X, maternal NM_005140.1 chrX:150911796 c.821G>A p.(Arg274His) 0.1757 0.1327 0.15 grandfather *300338 Present in maternal 15 CACNA1F X, maternal NM_005183.3 chrX:49083534 c.1174C>T p.(Arg392Trp) 0.0239 0.0095 0 grandfather* *300110 Present in two 20 PCDH11X X, maternal NM_032968.4 chrX:91090781 c.278G>A p.(Arg93His) 0.0439 0.0095 0 brothers *300246 21 ITPRIPL1 Paternal Present in sister NM_178495.5 chr2:96992802 c.457G>C p.(Glu153Gln) 0 0 0 21 ITPRIPL1 Maternal Present in sister NM_178495.5 chr2:96993332 c.987del p.(Trp329Cysfs*5) 0 0.0080 0 *An electroretinography excluded subclinical congenital stationary night blindness type II in the grandfather.

158 Novel genetic causes for CVI

159 Chapter 3.1

Supplementary Table S3: Classification of validated and segregating variants included based on the stringent filtering, part 1 S allele frequency (%) egregation in anamnestically egregation V Intramural allele frequency (%) Intramural Row number Row Patient Gene Inheritance S family members healthy number accession R efseq reference O MIM database numbers position ( H g 19) Chromosomal D NA change change Protein db SN P allele frequency (%) E

1 1 DLG4 De novo NP NM_001365.3 *602887 chr17:7107520 c.277dup p.(Tyr93Leufs*2) 0 0 0 2 2 NGLY1 Paternal & maternal NP NM_018297.3 *610661 chr3:25775422 c.1201A>T p.(Arg401*) 0 0.0231 0.08 3 3 HSPG2 Paternal Present in affected sister NM_001291860.1 *142461 chr1:22211048 c.1630C>T p.Arg544Cys 0 0 0.02 4 3 HSPG2 Maternal Present in affected sister NM_001291860.1 *142461 chr1:22206977 c.2077G>A p.Val693Met 0.1829 0.2466 0.6 5 3 TENM3 Paternal & maternal Homozygous present in NM_001080477.1 *610083 chr4:183601761 c.1705G>A p.Gly569Ser 0.87 0.0493 0.06 affected sister 6 6 SLC35A2 De novo NP NM_005660.1 *314375 chrX:48762386 c.800A>G p.(Tyr267Cys) 0 0 0 7 6 SRP72 De novo NP NM_006947.3 *602122 chr4:57356532 c.1354G>C p.Glu452Gln 0 0 0 8 7 AHDC1 De novo NP NM_001029882.2 *615790 chr1:27877225 c.1402dup p.(Cys468Leufs*49) 0 0 0 9 8 ARHGEF10L De novo NP NM_018125.3 *612494 chr1:18023681 c.3646G>A p.(Asp1216Asn) 0 0 0 10 8 GABRB2 De novo NP NM_021911.2 *600232 chr5:160761837 c.754C>G p.(Pro252Ala) 0 0 0 11 9 AMOT De novo NP NM_001113490.1 *300410 chrX:112035060 c.1926G>C p.Gln642His 0 0 0 12 10 OR52M1 De novo NP NM_001004137.1 - chr11:4566479 c.59C>T p.(Pro20Leu) 0 0 0 13 10 UHMK1 De novo NP NM_175866.4 *608849 chr1:162492294 c.1214C>T p.(Pro405Leu) 0 0 0 14 11 SLC25A16 De novo NP NM_152707.3 *139080 chr10:70246950 c.793C>T p.(Arg265Cys) 0 0 0 15 11 DCAF6 Paternal & maternal NP NM_001198956.1 *610494 chr1:168014447 c.2240G>A p.(Arg747Gln) 0.0471 0.0308 0.15 16 11 PHKB Paternal & maternal NP NM_000293.2 *172490 chr16:47536996 c.400G>A p.(Asp134Asn) 0.1115 0.1463 0.61 17 11 GRIN1 Paternal & maternal NP NM_007327.3 *138249 chr9:140051128 c.679G>C p.(Asp248His) 0 0 0 18 12 PGAP1 Maternal Not present in sister NM_024989.3 *611655 chr2:197761857 c.921_925del p.(Lys308Asnfs*25) 0 0 0 19 12 PGAP1 Paternal Heterozygous in sister NM_024989.3 *611655 chr2:197784746 c.274_276del p.(Pro92del) 0 0 0 20 13 NR2F1 De novo NP NM_005654.4 *132890 chr5:92921073 c.344G>C p.(Arg115Pro) 0 0 0

160 Novel genetic causes for CVI

1 1 DLG4 De novo NP NM_001365.3 *602887 chr17:7107520 c.277dup p.(Tyr93Leufs*2) 0 0 0 2 2 NGLY1 Paternal & maternal NP NM_018297.3 *610661 chr3:25775422 c.1201A>T p.(Arg401*) 0 0.0231 0.08 3 3 HSPG2 Paternal Present in affected sister NM_001291860.1 *142461 chr1:22211048 c.1630C>T p.Arg544Cys 0 0 0.02 4 3 HSPG2 Maternal Present in affected sister NM_001291860.1 *142461 chr1:22206977 c.2077G>A p.Val693Met 0.1829 0.2466 0.6 5 3 TENM3 Paternal & maternal Homozygous present in NM_001080477.1 *610083 chr4:183601761 c.1705G>A p.Gly569Ser 0.87 0.0493 0.06 affected sister 6 6 SLC35A2 De novo NP NM_005660.1 *314375 chrX:48762386 c.800A>G p.(Tyr267Cys) 0 0 0 7 6 SRP72 De novo NP NM_006947.3 *602122 chr4:57356532 c.1354G>C p.Glu452Gln 0 0 0 8 7 AHDC1 De novo NP NM_001029882.2 *615790 chr1:27877225 c.1402dup p.(Cys468Leufs*49) 0 0 0 Chapter 3.1 9 8 ARHGEF10L De novo NP NM_018125.3 *612494 chr1:18023681 c.3646G>A p.(Asp1216Asn) 0 0 0 10 8 GABRB2 De novo NP NM_021911.2 *600232 chr5:160761837 c.754C>G p.(Pro252Ala) 0 0 0 11 9 AMOT De novo NP NM_001113490.1 *300410 chrX:112035060 c.1926G>C p.Gln642His 0 0 0 12 10 OR52M1 De novo NP NM_001004137.1 - chr11:4566479 c.59C>T p.(Pro20Leu) 0 0 0 13 10 UHMK1 De novo NP NM_175866.4 *608849 chr1:162492294 c.1214C>T p.(Pro405Leu) 0 0 0 14 11 SLC25A16 De novo NP NM_152707.3 *139080 chr10:70246950 c.793C>T p.(Arg265Cys) 0 0 0 15 11 DCAF6 Paternal & maternal NP NM_001198956.1 *610494 chr1:168014447 c.2240G>A p.(Arg747Gln) 0.0471 0.0308 0.15 16 11 PHKB Paternal & maternal NP NM_000293.2 *172490 chr16:47536996 c.400G>A p.(Asp134Asn) 0.1115 0.1463 0.61 17 11 GRIN1 Paternal & maternal NP NM_007327.3 *138249 chr9:140051128 c.679G>C p.(Asp248His) 0 0 0 18 12 PGAP1 Maternal Not present in sister NM_024989.3 *611655 chr2:197761857 c.921_925del p.(Lys308Asnfs*25) 0 0 0 19 12 PGAP1 Paternal Heterozygous in sister NM_024989.3 *611655 chr2:197784746 c.274_276del p.(Pro92del) 0 0 0 20 13 NR2F1 De novo NP NM_005654.4 *132890 chr5:92921073 c.344G>C p.(Arg115Pro) 0 0 0

161 Chapter 3.1

Supplementary Table S3: Classification of validated and segregating variants included based on the stringent filtering, part 1 (continued) C truncating allel frequency C truncating allel frequency C truncating nalysis of mouse ontology nalysis Row number Row E x A per gene (%) phenotype O MIM related inheritance Corresponding type mutation and pattern, phenotype gene CVI-associated Known (disruptive/ of mutation N ature bepredicted pathogenic) to >3.5) Genomic (phyloP location pattern E xpression of gene ontology E nrichment term A Classification* E x A per gene (%) phenotype O MIM related

1 0.01 - - D C B G M Candidate 0.01 - 2 0.05 #615273 + K D NA NA NA NA Known 0.05 #615273 3 NA #224410, #255800 - - P - NA NA NA Unlikely NA #224410, #255800 4 NA #224410, #255800 - - P - NA NA NA Unlikely NA #224410, #255800 5 NA #615145 - - P C NA NA NA Unlikely NA #615145 6 NA #300896 + - P C NA NA NA Candidate NA #300896 7 NA #614675 - - P C NA NA NA Unlikely NA #614675 8 0 #615829 + K D NA NA NA NA Known 0 #615829 9 NA - - P C B - - Candidate NA - 10 NA - - P C B G M Candidate NA - 11 NA - - D (splice effect) NA B G M Candidate NA - 12 NA - - P - - - - Unlikely NA - 13 NA - - P C B G - Candidate NA - 14 NA - - P C - G - Candidate NA - 15 NA - - P - B G - Candidate NA - 16 NA #261750 - - P C NA NA NA Unlikely NA #261750 17 NA #614254 -§ - P C B - - Candidate NA #614254 18 0.02 #615802 + K D NA NA NA NA Known 0.02 #615802 19 0.02 #615802 + K - NA NA NA NA Known 0.02 #615802 20 NA #615722 + K P C NA NA NA Known NA #615722

162 Novel genetic causes for CVI

1 0.01 - - D C B G M Candidate 0.01 - 2 0.05 #615273 + K D NA NA NA NA Known 0.05 #615273 3 NA #224410, #255800 - - P - NA NA NA Unlikely NA #224410, #255800 4 NA #224410, #255800 - - P - NA NA NA Unlikely NA #224410, #255800 5 NA #615145 - - P C NA NA NA Unlikely NA #615145 6 NA #300896 + - P C NA NA NA Candidate NA #300896 7 NA #614675 - - P C NA NA NA Unlikely NA #614675 8 0 #615829 + K D NA NA NA NA Known 0 #615829 Chapter 3.1 9 NA - - P C B - - Candidate NA - 10 NA - - P C B G M Candidate NA - 11 NA - - D (splice effect) NA B G M Candidate NA - 12 NA - - P - - - - Unlikely NA - 13 NA - - P C B G - Candidate NA - 14 NA - - P C - G - Candidate NA - 15 NA - - P - B G - Candidate NA - 16 NA #261750 - - P C NA NA NA Unlikely NA #261750 17 NA #614254 -§ - P C B - - Candidate NA #614254 18 0.02 #615802 + K D NA NA NA NA Known 0.02 #615802 19 0.02 #615802 + K - NA NA NA NA Known 0.02 #615802 20 NA #615722 + K P C NA NA NA Known NA #615722

163 Chapter 3.1

Supplementary Table S3: Classification of validated and segregating variants included based on the stringent filtering, part 2 S allele frequency (%) egregation in anamnestically egregation V Intramural allele frequency (%) Intramural Row number Row Patient Gene Inheritance S family members healthy number accession R efseq reference O MIM database numbers position ( H g 19) Chromosomal D NA change change Protein db SN P allele frequency (%) E

21 13 NHLRC2 Maternal NP NM_198514.3 - chr10:115639414 c.869T>C p.(Ile290Thr) 0 0.0077 0 22 13 NHLRC2 Paternal NP NM_198514.3 - chr10:115663307 c.1516del p.(Thr506Glnfs*7) 0 0 0 23 13 OOSP2 Maternal NP NM_173801.3 - chr11:59812142 c.244-2A>G p.? 0 0 0 24 13 OOSP2 Paternal NP NM_173801.3 - chr11:59814500 c.431C>T p.(Thr144Ile) 0 0 0 25 15 RAB11FIP1 De novo NP NM_001002814.2 *608737 chr8:37729253 c.3067del p.(Leu1023Trpfs*17) 0 0 0 26 15 XKRX X, maternal Present in maternal NM_212559.2 *300684 chrX:100183023 c.271G>A p.(Asp91Asn) 0 0 0 grandmother 27 17 KCTD19 De novo NP NM_001100915.1 - chr16:67354577 c.215C>A p.(Thr72Asn) 0 0 0 28 17 SOX5 De novo NP NM_006940.4 *604975 chr12:23689544 c.1831C>G p.(Arg611Gly) 0 0 0 29 19 GRIN2B De novo NP NM_000834.3 *138252 chr12:13724844 c.2065G>A p.(Gly689Ser) 0 0 0 30 19 ZFP30 De novo NP NM_014898.2 - chr19:38126712 c.730T>C p.(Cys244Arg) 0 0 0 31 20 ATP6V1A De novo NP NM_001690.3 *607027 chr3:113508639 c.940A>G p.(Asn314Asp) 0 0 0 32 20 UFSP2 De novo NP NM_018359.3 *611482 chr4:186321583 c.1373A>G p.(Tyr458Cys) 0 0 0 33 22 RERE De novo NP NM_012102.3 *605226 chr1:8418302 c.4293C>A p.(His1431Gln) 0 0 0 34 22 SLC1A1† De novo NP NM_004170.5 *133550 chr9:4583108 c.1264G>A p.(Val422Met) 0 0 0 35 22 SYNE1 Maternal NP NM_182961.3 *608441 chr6:152476161 c.23995C>T p.(Arg7999*) 0 0 0 36 22 SYNE1 Paternal NP NM_182961.3 *608441 chr6:152614761 c.17974C>G p.(Pro5992Ala) 0.09 0 0.08 37 23 NR2F1 De novo NP NM_005654.4 *132890 chr5:92921068 c.339C>A p.(Ser113Arg) 0 0 0 38 23 ACP6 Paternal & NP NM_016361.3 *611471 chr1:147131611 c.379G>A p.(Val127Met) 0.1033 0.0846 0.23 maternal 39 24 KCNQ3 De novo NP NM_004519.3 *602232 chr8:133192493 c.688C>T p.(Arg230Cys) 0 0 0 40 25 APOPT1 De novo NP NM_032374.3 - chr14:104040507 c.424G>A p.(Gly142Ser) 0 0 0

164 Novel genetic causes for CVI

28 17 SOX5 De novo NP NM_006940.4 *604975 chr12:23689544 c.1831C>G p.(Arg611Gly) 0 0 0 Chapter 3.1 29 19 GRIN2B De novo NP NM_000834.3 *138252 chr12:13724844 c.2065G>A p.(Gly689Ser) 0 0 0 30 19 ZFP30 De novo NP NM_014898.2 - chr19:38126712 c.730T>C p.(Cys244Arg) 0 0 0 31 20 ATP6V1A De novo NP NM_001690.3 *607027 chr3:113508639 c.940A>G p.(Asn314Asp) 0 0 0 32 20 UFSP2 De novo NP NM_018359.3 *611482 chr4:186321583 c.1373A>G p.(Tyr458Cys) 0 0 0 33 22 RERE De novo NP NM_012102.3 *605226 chr1:8418302 c.4293C>A p.(His1431Gln) 0 0 0 34 22 SLC1A1† De novo NP NM_004170.5 *133550 chr9:4583108 c.1264G>A p.(Val422Met) 0 0 0 35 22 SYNE1 Maternal NP NM_182961.3 *608441 chr6:152476161 c.23995C>T p.(Arg7999*) 0 0 0 36 22 SYNE1 Paternal NP NM_182961.3 *608441 chr6:152614761 c.17974C>G p.(Pro5992Ala) 0.09 0 0.08 37 23 NR2F1 De novo NP NM_005654.4 *132890 chr5:92921068 c.339C>A p.(Ser113Arg) 0 0 0 38 23 ACP6 Paternal & NP NM_016361.3 *611471 chr1:147131611 c.379G>A p.(Val127Met) 0.1033 0.0846 0.23 maternal 39 24 KCNQ3 De novo NP NM_004519.3 *602232 chr8:133192493 c.688C>T p.(Arg230Cys) 0 0 0 40 25 APOPT1 De novo NP NM_032374.3 - chr14:104040507 c.424G>A p.(Gly142Ser) 0 0 0

165 Chapter 3.1

Supplementary Table S3: Classification of validated and segregating variants included based on the stringent filtering, part 2 (continued) C truncating allel frequency C truncating allel frequency C truncating nalysis of mouse ontology nalysis Row number Row E x A per gene (%) phenotype O MIM related inheritance Corresponding type mutation and pattern, phenotype gene CVI-associated Known (disruptive/ of mutation N ature bepredicted pathogenic) to >3.5) Genomic (phyloP location pattern E xpression of gene ontology E nrichment term A Classification* E x A per gene (%) phenotype O MIM related

21 NA - - - - B - - Unlikely NA - 22 0.02 - - D NA B - - Unlikely# 0.02 - 23 0.02 - - D NA - - - Unlikely 0.02 - 24 NA ------Unlikely NA - 25 0.06 - - D - - - - Unlikely 0.06 - 26 NA - - P C - - - Unlikely NA - 27 NA - - P C u G - Candidate NA - 28 NA - - P C B G M Candidate NA - 29 NA #613970 + - P C NA NA NA Candidate NA #613970 30 NA - - P C B - - Candidate NA - 31 NA - - P C B G - Candidate NA - 32 NA - - P C B - - Candidate NA - 33 NA - - P - B G M Candidate NA - 34 NA - - P C B G M Candidate NA - 35 0.35 #612998, #610743 - - D NA NA NA NA Unlikely 0.35 #612998, #610743 36 NA #612998, #610743 - - - - NA NA NA Unlikely NA #612998, #610743 37 NA #615722 + K P - NA NA NA Known NA #615722 38 NA - - P - B G - Candidate NA - 39 NA #121201 + - P - NA NA NA Candidate NA #121201 40 NA #220110 -‡ - P C NA NA NA Unlikely NA #220110

Supplementary Table S3: Classification of validated and segregating variants included based on the stringent (legend) B: Brain expressed. C: Evolutionary conserved genomic base (PhyloP>3.5). D: Disruptive mutation. G: GO- term enrichment. K: Known CVI-associated gene. M: Mouse phenotypic overlap. NA: Not assessed (irrel- evant due to mutation type/known ID gene). NP= Not performed. P: Predicted pathogenic (≥2 software programs). u: Unknown. -: Not. *See method section for classification process. †Mosaic mutation. ‡Phe- notype did not match and no second mutation was present (autosomal recessive disorder). §Inheritance does not fit with the reported OMIM disease (de novo autosomal dominant), this variant is further classified according to de Ligt and Gilissen et al, see result section. #Second mutation did not fulfilled the criteria.

166 Novel genetic causes for CVI

21 NA - - - - B - - Unlikely NA - 22 0.02 - - D NA B - - Unlikely# 0.02 - 23 0.02 - - D NA - - - Unlikely 0.02 - 24 NA ------Unlikely NA - 25 0.06 - - D - - - - Unlikely 0.06 - 26 NA - - P C - - - Unlikely NA - 27 NA - - P C u G - Candidate NA - 28 NA - - P C B G M Candidate NA - Chapter 3.1 29 NA #613970 + - P C NA NA NA Candidate NA #613970 30 NA - - P C B - - Candidate NA - 31 NA - - P C B G - Candidate NA - 32 NA - - P C B - - Candidate NA - 33 NA - - P - B G M Candidate NA - 34 NA - - P C B G M Candidate NA - 35 0.35 #612998, #610743 - - D NA NA NA NA Unlikely 0.35 #612998, #610743 36 NA #612998, #610743 - - - - NA NA NA Unlikely NA #612998, #610743 37 NA #615722 + K P - NA NA NA Known NA #615722 38 NA - - P - B G - Candidate NA - 39 NA #121201 + - P - NA NA NA Candidate NA #121201 40 NA #220110 -‡ - P C NA NA NA Unlikely NA #220110

167 Chapter 3.1

Supplementary Table S4: Classification of validated and segregating variants excluded based on the stringent filtering S allele frequency (%) egregation in anamnestically egregation V Row number Row Patient Gene Inheritance S family members healthy number accession R efseq reference O MIM database numbers position ( H g 19) Chromosomal D NA change change Protein db SN P allele frequency (%) E

1 2 AKAP9 De novo NP NM_005751.4 *604001 chr7:91694570 c.6005_6008del p.(Met2002Argfs*4) 0 0 2 2 KAL1 X, maternal NP NM_000216.2 *300836 chrX:8503715 c.1759G>T p.(Val587Leu) 0.2417 0.2659 3 10 PPFIA4† De novo NP NM_015053.1 *603145 chr1:203025958 c.769C>T p.(Gln257*) 0 0 4 10 MAP3K15 X, maternal NP NM_001001671.3 *300820 chrX:19398315 c.2512G>A p.(Gly838Ser) 0.2585 0.2562 5 11 SPTBN5 Paternal & maternal NP NM_016642.3 *605916 chr15:42147078 c.9520dup p.(Arg3174Profs*18) 0 0 6 11 FAM166B Paternal NP NM_001164310.1 - chr9:35562084 c.745_748dup p.(Phe250*) 0 0.0528 7 11 FAM166B Maternal NP NM_001164310.1 - chr9:35563190 c.259A>G p.(Ser87Gly) 0 0.0871 8 13 POF1B X, maternal NP NM_024921.3 *300603 chrX:84569452 c.943C>T p.(Arg315Cys) 0.0883 0.0663 9 17 TRIOBP De novo NP NM_001039141.2 *609761 chr22:38121122 c.2559del p.(Thr854Hisfs*25) 0 0 10 18 MUT De novo NP NM_000255.3 *609058 chr6:49419396 c.1115T>C p.(Ile372Thr) 0.1102 0.0462 11 25 SLC6A13 De novo NP NM_016615.4 *615097 chr12:369056 c.163G>A p.(Val55Ile) 0.022 0.0154 12 25 ALAS2 X, maternal Not present in maternal NM_001037968.3 *301300 chrX:55054238 c.3G>A p.(Met1?) 0 0.3549 uncle or grandfather

168 Novel genetic causes for CVI

1 2 AKAP9 De novo NP NM_005751.4 *604001 chr7:91694570 c.6005_6008del p.(Met2002Argfs*4) 0 0 2 2 KAL1 X, maternal NP NM_000216.2 *300836 chrX:8503715 c.1759G>T p.(Val587Leu) 0.2417 0.2659 3 10 PPFIA4† De novo NP NM_015053.1 *603145 chr1:203025958 c.769C>T p.(Gln257*) 0 0 4 10 MAP3K15 X, maternal NP NM_001001671.3 *300820 chrX:19398315 c.2512G>A p.(Gly838Ser) 0.2585 0.2562 5 11 SPTBN5 Paternal & maternal NP NM_016642.3 *605916 chr15:42147078 c.9520dup p.(Arg3174Profs*18) 0 0 6 11 FAM166B Paternal NP NM_001164310.1 - chr9:35562084 c.745_748dup p.(Phe250*) 0 0.0528 7 11 FAM166B Maternal NP NM_001164310.1 - chr9:35563190 c.259A>G p.(Ser87Gly) 0 0.0871 8 13 POF1B X, maternal NP NM_024921.3 *300603 chrX:84569452 c.943C>T p.(Arg315Cys) 0.0883 0.0663 Chapter 3.1 9 17 TRIOBP De novo NP NM_001039141.2 *609761 chr22:38121122 c.2559del p.(Thr854Hisfs*25) 0 0 10 18 MUT De novo NP NM_000255.3 *609058 chr6:49419396 c.1115T>C p.(Ile372Thr) 0.1102 0.0462 11 25 SLC6A13 De novo NP NM_016615.4 *615097 chr12:369056 c.163G>A p.(Val55Ile) 0.022 0.0154 12 25 ALAS2 X, maternal Not present in maternal NM_001037968.3 *301300 chrX:55054238 c.3G>A p.(Met1?) 0 0.3549 uncle or grandfather

169 Chapter 3.1

Supplementary Table S4: Classification of validated and segregating variants excluded based on the stringent filtering (continued) C truncating allel frequency C truncating nalysis of mouse ontology nalysis Row number Row allele frequency (%) Intramural E x A per gene (%) phenotype O MIM related inheritance Corresponding type mutation and pattern, phenotype gene CVI-associated Known (disruptive/ of mutation N ature bepredicted pathogenic) to >3.5) Genomic (phyloP location pattern E xpression of gene ontology E nrichment term A Classification*

1 0 0.23 #611820 - - D NA NA NA NA Unlikely 2 0.04 NA #308700 -ǁ - P C NA NA NA Unlikelyǁ 3 0 26.65 - - D NA u G - Candidate 4 0.84 NA - - P C - G - Candidate 5 0.77 4.72 - - D NA - G - Candidate 6 0.08 15.48 - - D NA u - - Unlikely 7 0.08 NA - - - - u - - Unlikely 8 0 NA #300604 - - P C NA NA NA Unlikely 9 0 2.13 #609823 -‡ - D NA NA NA NA Unlikely 10 0.08 NA #251000 -‡ - P C NA NA NA Unlikely 11 0.23 NA - - P - - G - Unlikely 12 0.38 0 #300751, #300752 - - D NA NA NA NA Unlikely

Supplementary Table S4: Classification of validated and segregating variants excluded based on the stringent filtering (legend) B: Brain expressed. C: Evolutionary conserved genomic base (PhyloP>3.5). D: Disruptive mutation. G: GO- term enrichment. K: Known CVI-associated gene. M: Mouse phenotypic overlap. NA: Not assessed (irrel- evant due to mutation type/known ID gene). NP= Not performed. P: Predicted pathogenic (≥2 software programs). u: Unknown. -: Not.*See method section for classification process. †Mosaic mutation. ‡Pheno- type did not match and no second mutation was present (autosomal recessive disorder). ǁThe phenotypic spectrum of KAL1 is well known, and no CVI is reported, therefore we classified this variant as unlikely to be associated with CVI, although some of the other features of the patient might be ascribed to this variant.

170 Novel genetic causes for CVI

1 0 0.23 #611820 - - D NA NA NA NA Unlikely 2 0.04 NA #308700 -ǁ - P C NA NA NA Unlikelyǁ 3 0 26.65 - - D NA u G - Candidate 4 0.84 NA - - P C - G - Candidate 5 0.77 4.72 - - D NA - G - Candidate 6 0.08 15.48 - - D NA u - - Unlikely 7 0.08 NA - - - - u - - Unlikely 8 0 NA #300604 - - P C NA NA NA Unlikely Chapter 3.1 9 0 2.13 #609823 -‡ - D NA NA NA NA Unlikely 10 0.08 NA #251000 -‡ - P C NA NA NA Unlikely 11 0.23 NA - - P - - G - Unlikely 12 0.38 0 #300751, #300752 - - D NA NA NA NA Unlikely

171

3.2

Cerebral visual impairment and intellectual disability caused by PGAP1 variants

Daniëlle G.M. Bosch, F. Nienke Boonstra, Taroh Kinoshita, Shalini Jhangiani, Joep de Ligt, Frans P.M. Cremers, James R. Lupski, Yoshiko Murakami, Bert B. A. de Vries

Eur J Hum Genet 2015; 23: 1689-1693. Chapter 3.2

Abstract

Homozygous variants in PGAP1 (post-GPI attachment to proteins 1) have recently been identified in two families with developmental delay, seizures and/or spasticity. PGAP1 is a member of the glycosylphosphatidylinositol -anchor biosynthesis and remodelling pathway and defects in this pathway are a subclass of congenital disorders of glycosylation. Here we performed whole exome sequencing in an individual with cerebral visual impairment (CVI), intellectual disability (ID), and factor XII deficiency and revealed compound heterozygous variants in PGAP1, c.274_276del (p.(Pro92del)) and c.921_925del (p.(Lys308Asnfs*25)). Subsequently, PGAP1-deficient Chinese hamster ovary (CHO)-cell lines were transfected with either mutant or wild-type constructs and their sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment was measured. The mutant constructs could not rescue the PGAP1-deficient CHO cell lines resistance to PI-PLC treatment. In addition, lymphoblastoid cell lines (LCLs) of the affected individual showed no sensitivity to PI-PLC treatment, whereas the LCLs of the heterozygous carrier parents were partially resistant. In conclusion, we report novel PGAP1 variants in a boy with CVI and ID and a proven functional loss of PGAP1 and show, to our knowledge, for the first time this genetic association with CVI.

174 CVI and ID caused by PGAP1 variants

Introduction

Cerebral visual impairment (CVI) accounts for 27% of the visual impairment in children, and is due to a disorder in projection and/or interpretation of the visual input in the brain.47,53,55 Acquired damages, for example due to preterm birth, are well known causes of CVI, whereas genetic factors are largely unidentified.250 Several chromosomal aberrations have been associated with CVI, such as Phelan-McDermid syndrome (22q13.3 deletion) and 1p36 microdeletion syndrome.251 Moreover, single gene disorders can be implicated in CVI, and, recently, we identified de novo variants in NR2F1 as a cause of CVI.191 In addition, in several congenital disorders of glycosylation (CDG type 1a, type 1q, and type 1v) CVI has been reported.126,127,250,259 Glycosylation disorders are caused by a defect in the glycosylation of glyco- proteins and glycolipids, and one subclass is the defect in glycosylphosphatidylinositol-(GPI-) anchor glycosylation.309 GPI anchor many cell-surface proteins with various functions, so called GPI-anchored proteins (GPI-APs), to the membrane of eukaryotic cells.310-312 GPI is synthesized in the endoplasmic reticulum, transferred to the proteins, and remodelled. During the biosynthesis of GPI anchors an acyl chain is linked to the inositol. This acyl-chain is a transient structure and is necessary for efficient completion of later steps in GPI biosynthesis. After the attachment of GPI to the protein, this acyl-chain is removed by PGAP1 (post-GPI attachment to proteins 1), the first step of the remodelling phase.313 Subsequently, the GPI-anchor is transported from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus and further Chapter 3.2 remodelled. When the inositol-linked acyl-chain is not removed this transport is delayed,313 but the presence of this acyl-chain does not affect the cell surface expression of GPI-APs.270 Several genes known to be involved in the GPI-anchor biosynthesis (PIGA, PIGL, PIGN, PIGT, PIGV, PIGO, PIGW and PIGQ) and modelling (PGAP2 and PGAP3) are implicated in X-linked and autosomal recessive intellectual disability disorders.169,267,268,314-321 Additional features such as seizures, congenital abnormalities and facial dysmorphisms are commonly present. Moreover, CVI has been reported in individuals with PIGA, PIGN and PIGT variants, suggesting that the GPI-anchor biosynthesis is important in the development of the visual areas of the brain.266-269 Recently, two families with developmental delay, seizures and/or spasticity were reported with homozygous variants in PGAP1.270,322 However, PGAP1 variants have not to date been associated with CVI. Here, we report an individual with CVI and intellectual disability (ID) and variants in PGAP1, thereby showing the association between PGAP1 and CVI.

175 Chapter 3.2

Individual and methods

Case report The boy was the second child of healthy unrelated parents. He was born after a normal pregnancy by a planned caesarean section, because of a difficult delivery of the previous child. His birth weight was 3550 gram (70th centile) at a gestational age of 38 weeks and Apgar scores were 9 and 10, after 1 and 5 min, respectively. During the neonatal period he was hypotonic and there were feeding difficulties. His development was delayed as he started walking and speaking around the age of 2-2½ years. At the age of 8 years 3 months, his total IQ was 49 (Wechsler Intelligence Scale for Children-III). He was very social in his behaviour. There were no signs of epilepsy and an MRI of the brain was normal at age 5 years. Hearing was normal, but ophthalmological examination at 7 years revealed CVI with a visual acuity of 0.3 (Landolt C). He had strabismus divergence and nystagmus. Slit lamp examination and funduscopy were normal. He looked only shortly at a target and he had more difficulties with recognition of objects in clutter (crowding) than was expected for his age. He had mild asthma and his APTT was prolonged due to a factor XII deficiency (factor XII activity <1%). His mother and sister also had decreased factor XII activity (40% and 22%, respectively), with a normal APTT. There were no signs of immunodeficiency. Physical examination at 7 years 10 months showed a height of 131 cm (50th centile) and head circumference of 52.5 cm (50th centile). Facial dysmorphisms consisted of upward slanting palpebral fissures, deep-set eyes, large ear lobes and prominent helices and antihelices (Figure 1). His teeth showed extra mamelons and diminished enamel was noted. Previous genetic studies, consisting of FMR1 gene, AIP gene, array CGH and metabolic studies (including transferrin glycosylation assay) showed normal results. This study was approved by the Ethics Committee of the Radboud university medical center (Commissie Mensgebonden Onderzoek, regio Arnhem-Nijmegen), and written informed consent was obtained.

Whole-exome sequencing WES in this individual (CVI10) was performed as part of a larger study to identify the genetic causes of CVI.191 WES was performed by the Baylor-Hopkins Center for Mendelian Genomics, using methods reported previously (Supplementary Material and Methods, Whole-exome sequencing).191,303 The results were analysed for de novo variants, homozygous variants, compound heterozygous variants and hemizygous variants as described in de Ligt et al.182 Truncating variants, splice site variants and missense variants predicted to be pathogenic were validated by Sanger sequencing in the affected subject and his parents. The variants identified have been submitted to the Leiden Open Variation Database LOVD database (http://databases.lovd.nl/, individual #00025011).

176 CVI and ID caused by PGAP1 variants

B Previously p.(Leu197del) c.1952+1G>T† reported:

N C

0 922

This study: p.(Pro92del) p.(Ala305Val)†† p.(Lys308Asnfs*25)

C Wild-type c.274_276del c.914C>T c.921_925del (p.(Pro92del)) (p.(Ala305Val)) (p.(Lys308Asnfs))

CD59 Chapter 3.2

DAF

uPAR

Isotype control PI-PLC - PI-PLC + Figure 1: Phenotype of the affected individual and the functional analysis of the variants A) Photographs of the affected individual, note the upward slanting palpebral fissures, deep set eyes, large ear lobes and prominent helices and antihelices. B) Schematic representation of PGAP1, containing transmembrane domains (in green). The positions of the variants identified in this study and in the previously reports are indicated. †For this variant the effect on amino acid sequence was not studied. ††Considered as a variant without functional impact. C) CHO cells deficient forPGAP1 were used to investigate the expected structural abnormalities of the GPI-anchors by testing the sensitivity of GPI-APs to PI-PLC. Wild-type PGAP1 and the construct containing the variant c.914C>T (p.(Ala305Val)) rescued the sensitivity for PI-PLC strongly suggesting that this variant is benign. The PGAP1 constructs containing the c.274_276del (p.(Pro92del)) and c.921_925del (p.(Lys308Asnfs*25)) variants did not increase the sensitivity for PI-PLC, suggesting that both are causal.

177 Chapter 3.2

Functional analysis using CHO cells PGAP1 deficient CHO cells (C10)313 were transiently transfected with wild type or mutant pMEFLAG-hPGAP1 by electroporation as reported previously (Supplementary Material and Methods, Functional analysis using CHO cells)270 Four days after transfection, cells were treated with or without phosphatidylinositol-specific phospholipase C (PI-PLC). Surface expression of GPI-APs was determined by staining cells with mouse anti-human CD59 (5H8), -human DAF (IA10), -hamster uPAR (5D6) antibodies and analysed by flow cytometry using Flowjo software (Tommy Digital Inc., Tokyo, Japan).

PI-PLC treatment and FACS analysis Heparin blood samples were collected from the affected individual and his unaffected parents and lymphoblastoid cell lines (LCLs) were generated (Supplementary Material and Methods, PI-PLC treatment and FACS analysis). Four days after transfection, cells were treated with or without PI-PLC as reported previously.270 Surface expression of GPI-APs was determined by staining cells with mouse anti-human CD59 (5H8), -human DAF (IA10), -human CD48 (BJ40) antibodies and analysed by flow cytometry using Flowjo software.

Results

WES WES in the affected individual and the parents was performed with an average coverage of 126x. After applying the above mentioned prioritizations step only two variants in PGAP1 remained, a paternal inherited chr2.hg19:g.197784746_197784748del; c.274_276del; p.(Pro92del) and a maternal inherited chr2.hg19:g.197761857_197761861del; c.921_925del; p.(Lys308Asnfs*25) (MIM 611655, RefSeq accession number NM_024989.3) (Supplementary Material, Supplementary Table S1). The variants were validated by Sanger sequencing, and an additional maternal inherited variant was identified, chr2.hg19:g.197761868C>T; c.914C>T; p.(Ala305Val), which was previously filtered out due to quality settings (Supplementary Mate- rial, Supplementary Figure S1). In addition, the exome results were screened for rare variants (<1% occurrence) in the coding regions of F12 (MIM 610619, RefSeq accession number NM_000505.3). A paternal inherited heterozygous variant was identified and validated with Sanger sequencing chr5.hg19:g.176831388G>A; c.827G>A; p.(Trp276*).

Functional analysis using CHO cells Under the presence of PI-PLC structurally normal GPI-APs, without inositol-linked acyl-chain, are cleaved from the cell membrane.323 CHO cells deficient forPGAP1 were used to investigate the expected structural abnormalities of the GPI-anchors by testing the sensitivity of GPI-APs to PI-PLC. Wild-type PGAP1 and the p.(Ala305Val) rescued the sensitivity for PI-PLC (Figure 1

178 CVI and ID caused by PGAP1 variants and Supplementary Material, Supplementary S2). However, transfection with empty vector, the p.(Pro92del) mutant or p.(Lys308Asnfs*25) mutant did not increase the sensitivity for PI- PLC (Supplementary Material, Supplementary Figure S3).

PI-PLC treatment and FACS analysis In addition, the LCLs of the affected individual and parents were analysed for PI-PLC resistance. The LCLs of the affected individual were completely resistant to PI-PLC treatment, whereas the LCLs of the parents were partially resistant (Supplementary Material, Supplementary Figure S4).

Discussion

Here we present an individual with CVI, ID, minor facial dysmorphisms and factor XII deficiency. WES revealed compound heterozygous variants in PGAP1, c.274_276del (p.(Pro92del)), c.914C>T (p.(Ala305Val)) and c.921_925del (p.(Lys308Asnfs*25)). To investigate the functional impact of the variants, PI-PLC sensitivity assay was performed in PGAP1 deficient CHO cells. The c.914C>T (p.(Ala305Val)) mutant rescued the PI-PLC-sensitivity in PGAP1 deficient CHO cells, similar to wild-type PGAP1. Therefore, this variant was considered to have no functional impact. The c.274_276del (p.(Pro92del)) and c.921_925del (p.(Lys308Asnfs*25)) mutant constructs, however, were not able to rescue the sensitivity, indicating a functional loss of the Chapter 3.2 cDNA constructs. In addition, the PI-PLC sensitivity was investigated in LCLs derived from the affected individual and parents. The LCLs of the affected individual were resistant to PI- PLC, indicating that the GPI-APs are structurally abnormal, whereas the parents showed a partial resistance. These results are in line with the reported family with PGAP1 variants by Murakami et al. The other family reported by Novarino et al. was not tested.270,322 Besides a developmental delay, the phenotype is variable. The reported siblings by Murakami et al. had severe developmental delay with seizures, stereotypic movements and brain atrophy (Table 1). The siblings reported by Novarino et al. had spasticity and structural brain abnormalities. In the individual described in this study, no seizures or brain abnormalities were present, but he had impaired vision and CVI. No ophthalmological examination was performed in the Murakami siblings. In the Novarino siblings no ‘ophthalmological signs’ were present, but whether complete ophthalmological examination had been performed remained unclear.

Depending on the genetic background of the mouse strain used, PGAP1 deficiency in mice could lead to various phenotypes, ranging from a normal structural brain to forebrain abnormalities, including holoprosencephaly.324-327 In the mutant phenotypically abnormal mice Wnt signalling and Nodal signaling were reported to be affected.325,327 However, the precise

179 Chapter 3.2

Table 1: phenotype of individuals with PGAP1 variants This study Murakami Murakami Novarino 1241- Novarino III-2 III-3 IV-3 1241-IV-4 Variant identified c.274_276del c.589_591del c.589_591del c.1952+1G>T and c.1952+1G>T and (Hg 19) (p.(Pro92del)) (p.(Leu197del)) (p.(Leu197del)) c.1952+1G>T c.1952+1G>T and c.921_925del and c.589_591del and c.589_591del (p.(Lys308Asnfs*25)) (p.(Leu197del)) (p.(Leu197del)) Gender M F M M M Birth weight 3550 g Normal Normal NA NA (70th centile) Gestational age 38 Normal Normal NA NA (weeks) Age at 7+10 4+5 2+9 6+6 0+9 investigations (years+months) Height 131 cm 96 cm Normal NA NA (50th centile) (25th centile) OFC 52.5 cm 46 cm 47 cm NA NA (50th centile) (<5th centile) (<5th centile) Delayed + + + + + development Walking 2+6 4+5 - - - independently (years+months) First words 2+6 - - NA NA (years+months) Hypotonia + + + NA NA Brain imaging Normal (MRI) Pronounced brain NP Prominent cortical Corpus callosum (MRI/CT) atrophy (CT) sulci & widened agnesis, vermis sylvian fissures hypoplasia, (MRI) defective myelination (MRI) Hearing Normal NP NP NA NA investigation Ophthalmo- CVI, strabismus, NP NP - - logical nystagmus examination Other Factor XII deficiency Stereotypic Stereotypic Spasticity Spasticity, abnormalities movements, movements distented seizures abdomen Dysmorphism Upward slanting Large ears, Large ears, NA NA palpebral fissures, deep flattenend nasal flattenend nasal set eyes, large ear lobes, bridge bridge prominent helices and antihelices, teeth showed extra mamelons with diminished enamel

NP, not performed; NA, not available.

180 CVI and ID caused by PGAP1 variants role of PGAP1 in the function and development of the brain and more specific of the visual system remains to be elucidated. One marker of CDG-syndromes can be the abnormal glycosylation of transferrin.328 How- ever, in the subclass of GPI-anchor glycosylation defects no abnormal transferrin is detect- able, making this marker unreliable in diagnosing GPI-anchor glycosylation defects, such as those caused by variants in PGAP1. One other feature of CDG syndromes is the deficiency of coagulation factors.328 However, factor XII deficiency has not been reported so far, neither has a direct functional link between factor XII and GPI-APs been made. Factor XII deficiency can be caused by variants in F12; a heterozygous variant can lead to an intermediated level of factor XII activity, whereas homozygous or compound heterozygous variants lead to almost no activity (<1%).329,330 The current individual had a factor XII activity <1%, and a paternal inherited loss of function variant in F12, c.827G>A (p.(Trp276*)), but his father had a normal factor XII activity. The mother had an intermediate factor XII activity, but no variant could be identified in her exome results. Whether the variants in PGAP1 are related to the factor XII deficiency is yet unclear. Two other GPI anchor-modelling proteins, PGAP2 and PGAP3, are implicated in hyperphosphatasia with mental retardation syndrome, also named Mabry syndrome.317-319 These individuals showed, in addition to ID, seizures, typical facial dysmorphisms and an increased alkaline phosphatase (ALP). This increase is due to diminished GPI-APs on the cell surface, resulting in less binding of ALP to the cell membrane and more ALP in the plasma.317-319,331,332 ALP was not measured in the PGAP1-affected individuals, but in PGAP1-deficient cells no Chapter 3.2 diminished cell surface expression of GPI-APs was measured, making elevated ALP levels less likely.270 In addition, the typical facial dysmorphisms of Mabry syndrome, consisting of apparent hypertelorism, long palpebral fissures, short nose with broad nasal bridge and tip and tented upper lip vermillion, were not present in the here presented individual. In conclusion, we identified novel PGAP1 variants in an intellectual disabled boy with a proven functional loss of PGAP1, and showed for the first time the genetic association with cerebral visual impairment. Additional affected individuals will be required to gain better insights into pathophysiology and acquire knowledge of the clinical spectrum of this novel disorder.

181 Chapter 3.2

Supplemental methods

Whole-exome sequencing (WES) WES was performed by the Baylor-Hopkins Center for Mendelian Genomics, using methods reported previously.191,303 In short, sequencing was performed on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA) and the sequence reads were mapped and aligned to the Human Genome Reference Assembly GRCh37/hg19 with the BCM-HGSC Mercury pipeline.333 Variant calling was performed with the Atlas2334 and SAMtools335 algorithms; variant annotation was performed with an intramurally developed annotation pipelin336 based on ANNOVAR337 and custom scripts to incorporate multiple databases to inform on identified variants. After quality filtering (variant reads >15%) local and global variants were excluded (>1% occurrence in intramural database, dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), Exome Vari- ant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (http://evs.gs.washington. edu/EVS/) or cohort frequency ≤2% (cohort of patients with various disorders and their healthy parents, N=200). The exonic nonsynonymous and canonical splice site variants were selected for further analysis. A de novo analysis was performed. Furthermore, the results were analysed for homozygous variants (>80% variant reads), compound heterozygous variants (two or more variants present in one gene) and hemizygous variants in males (X-chromosome, >80% variant reads). When the autosomal recessive variants were present at one allele the variants were excluded (based on the raw data, BAM-file, of the affected individual and his parents). Disrupting variants, consisting of frameshift, nonsense or splice site variants (assessed using Interac- tive Biosoftware Alamut version 2.3 rev2), and missense variants predicted to be pathogenic (majority vote of Polyphen-2, MutPred and SNP&GO were validated by Sanger sequencing in the affected subject and his parents (http://genetics.bwh.harvard.edu/pph2/, http:// mutpred.mutdb.org/, http://snps.path.uab.edu/snps-and-go/pages/method.html).304-306 For compound heterozygous variants at least one of the variants should be disrupting, or, when concerning missense variants, both variants should be predicted to be pathogenic.

Functional analysis using CHO cells pMEFLAG-hPGAP1 mutant bearing the variants of the affected individual were generated by site directed mutagenesis. PGAP1 deficient CHO cell (C10)313 were transiently transfected with wild type or mutant pMEFLAG-hPGAP1 by electroporation. Cells (107) were suspended in 0.4 ml of Opti-MEM and electroporated with 20 µg each of the plasmids at 260 V and 960 mF using a Gene Pulser. Four days after transfection, cells were treated with or without 10 unit/ml of phosphatidylinositol-specific phospholipase C (PI-PLC, Molecular probes, Eugene, OR)) for 1.5 h at 37oC. Surface expression of GPI-APs was determined by staining cells with mouse anti-human CD59 (5H8), -human DAF (IA10), -hamster uPAR (5D6) antibodies and

182 CVI and ID caused by PGAP1 variants each isotype IgG, followed by a PE-conjugated anti-mouse IgG antibody and analysed by flow cytometry using Flowjo software (Tommy Digital Inc., Tokyo, Japan).

PI-PLC treatment and FACS analysis Heparin blood samples were collected from the affected individual and his unaffected parents. Lymphoblastoid cell lines (LCLs) were generated and cultured in RPMI 164-R2405 (Sigma- Aldrich, St. Louis, United States of America) that was supplemented with 15% FCS-F7524, Pen/Strep-P4333 and HEPES-H0887 (Sigma-Aldrich, St. Louis, United States of America). The LCLs were transfected with empty pMEoriP vector or pMEoriPFLAG-humanPGAP1. Cells (56106) were suspended in 0.8 ml of Opti-MEM and electroporated with 20 µg each of the plasmids at 260 V and 960 mF using a Gene Pulser (Bio Rad, Hercules, CA). Four days after transfection, cells were treated with or without 10 unit/ml of PI-PLC for 1.5 h at 37oC. Surface expression of GPI-APs was determined by staining cells with mouse anti-human CD59 (5H8), -human DAF (IA10), -human CD48 (BJ40) antibodies and each isotype IgG followed by a PE- conjugated anti-mouse IgG antibody (BJ40, mouse IgG1 and IgG2a, and secondary antibody were purchased from BDBiosciences, Franklin Lakes, NJ) and analysed by flow cytometry (Cant II; BD Biosciences) using Flowjo software (Tommy Digital Inc., Tokyo, Japan). Chapter 3.2

183 Chapter 3.2

Supplemental table

Table S1: Characteristics of the identified variants Gene PGAP1 PGAP1 PGAP1 F12

RefSeq accession number NM_024989.3 NM_024989.3 NM_024989.3 NM_000505.3

Inheritance Paternal Maternal Maternal Paternal

Coding DNA change (Hg c.274_276del c.921_925del c.914C>T c. 827G>A 19)

Protein change p.(Pro92del) p.(Lys308Asnfs*25) p.(Ala305Val) p.(Trp276*)

dbSNP frequency 0 0 0 0

EVS 0 0 0 0

PhyloP NA NA 1.82 NA

Pholyphen2 (score) NA NA Benign NA

SNPs&Go (score) NA NA Neutral NA

MutPred NA NA 0.351 NA

NA, not available.

Supplemental figures

Patient

Father

Mother G G T A A T G C T T A A G T A T G T A T A C T T C C T G G T A A T G A T G C T G A T A C T A A A C A A G T A C C G T G G T G C Lys Pro Gly Ala Asp Ala Asp Thr Lys Gln Val Pro Trp Cys PGAP1 c.274_276del PGAP1 c.914C>T PGAP1 c.921_925del F12 c.827G>A p.(Pro92del) p.(Ala305Val) p.(Lys308Asnfs*25) p.(Trp276*)

Figure S1: Sanger sequences results of identified variants Paternal inherited variants in PGAP1 (NM_024989.3 , c.274_276del; p.(Pro92del)) and F12 (NM_000505.3, c.827G>A; p.(Trp276*)), and two maternal inherited variants in PGAP1 (c.914C>T; p.(Ala305Val) and c.921_925del; p.(Lys308Asnfs*25)) were validated by using Sanger sequencing.

184 CVI and ID caused by PGAP1 variants p.(Ala305Val) p.(Pro92del) Chapter 3.2 (NM_024989.3). The identified variant c.274_276del; p.(Pro92del) is indicated. B) isAmino indicated. acidconservap.(Pro92del) The identifiedc.274_276del;variant - PGAP1 (NM_024989.3). mino acid conservation of the identified variants of the identified mino acid conservation A Human Chimp Gorilla Orangutan White-tu ed-ear marmoset Rat Mouse Dog Chicken Tetraodon Fruity yeast Baker’s B Human Chimp Gorilla Orangutan White-tu ed-ear marmoset Rat Mouse Dog Chicken Tetraodon Fruity yeast Baker’s igure S 2: A igure (NM_024989.3). The identified variant c.914C>T; p.(Ala305Val) is indicated. p.(Ala305Val) c.914C>T; variant identified The 7 of PGAP1 (NM_024989.3). tion of exon A) A) Amino acid conservation of the distal part of 2 exon of F

185 Chapter 3.2

c.914C>T (p.(Ala305Val))/ c.274_276del c.914C>T c.921_925del c.921_925del Wild-type (p.(Pro92del)) (p.(Ala305Val)) (p.(Lys308Asnfs)) (p.(Lys308Asnfs)) Empty vector

CD59

DAF

uPAR

Isotype control PI-PLC - PI-PLC + figure s3: functional analysis of the identifi ed variants using Cho cells CHO cells defi cient for PGAP1 were used to investigate the expected structural abnormalities of the GPI anchors by testing the sensitivity of GPI-APs to PI-PLC. Wild-type PGAP1 and the c.914C>T (p.(Ala305Val)) rescued the sensitivity for PI-PLC. However, transfection with empty vector, the c.274_276del (p.(Pro92del)) mutant or c.921_925del (p.(Lys308Asnfs*25)) mutant did not increase the sensitivity for PI-PLC.

CD59 DAF CD48 CD59 DAF CD48

A ected

Mother

Father

Empty vector PGAP1 Isotype control PI-PLC - PI-PLC + figure s4: PI-PlC sensitivity in lCls the individual and his parents The LCLs of the aff ected individual were completely resistant to PI-PLC treatment, whereas the LCLs of the parents were partially resistant (Empty vector in left panels). After transfection of wild-type PGAP1 cDNA, sensitivity to PI-PLC was restored to the LCLs of the aff ected individual and the parents (PGAP1 in right panels), confi rming that the complete and partial resistance was due to defective PGAP1.

186

3.3

NR2F1 mutations cause optic atrophy with intellectual disability

Daniëlle G.M. Bosch,* F. Nienke Boonstra,*Claudia Gonzaga-Jauregui,* Mafei Xu, Joep de Ligt, Shalini Jhangiani, Wojciech Wiszniewski, Donna M. Muzny, Helger G. Yntema, Rolph Pfundt, Lisenka E.L.M. Vissers, Liesbeth Spruijt, Ellen A.W. Blokland, Chun-An Chen, Baylor-Hopkins Center for Mendelian Genomics, Richard A. Lewis, Sophia Y. Tsai, Richard A. Gibbs, Ming-Jer Tsai, James R. Lupski, Huda Y. Zoghbi, Frans P.M. Cremers,† Bert B. A. de Vries,† Christian P. Schaaf†

Am J Hum Genet 2014; 94: 303-309.

*These authors contributed equally to this work. †These authors contributed equally to this work and are co-senior authors. Chapter 3.3

Abstract

Optic nerve atrophy and hypoplasia can be primary disorders or can result from trans-synaptic degeneration arising from cerebral visual impairment (CVI). Here we report six individuals with CVI and/or optic nerve abnormalities, born after an uneventful pregnancy and delivery, who have either de novo heterozygous missense mutations in NR2F1, also known as COUP- TFI, or deletions encompassing NR2F1. All affected individuals show mild to moderate intellectual impairment. NR2F1 encodes a nuclear receptor protein that regulates transcription. A reporter assay showed that missense mutations in the zinc-finger DNA-binding domain and the putative ligand-binding domain decrease NR2F1 transcriptional activity. These findings indicate that NR2F1 plays an important role in the neurodevelopment of the visual system and that its disruption can lead to optic atrophy with intellectual disability.

190 NR2F1 mutations cause optic atrophy with ID

Report

Optic nerve abnormalities are increasingly recognized as a cause of poor vision. The most frequently identified abnormalities consist of optic nerve atrophy or hypoplasia, due to either genetic or acquired causes.55 Optic nerve hypoplasia is often reported as part of a syndrome, for example, in conjunction with structural malformations of the brain or hypopituitarism, as observed in SOX2-related disorders [MIM 184429],338 or combined with anterior segmental defects of the eye, such as iris coloboma in PAX6-related diseases [MIM 307108].339 Optic nerve atrophy, however, can be part of a progressive syndromic or nonsyndromic disorder. The genetic defects underlying many syndromic forms of optic atrophy are known, but the molecular bases of nonsyndromic forms have been elucidated in only a few instances: mutations in OPA1 [MIM 605290] and OPA3 [MIM 606580] (autosomal dominant inheritance),340,341 in TMEM126A [MIM 612988] (autosomal recessive inheritance),342 and in mitochondrial DNA as part of the Leber Hereditary Optic Neuropathy (LHON) [MIM 535000].343 Both optic atrophy and hypoplasia can be caused by trans-synaptic degeneration as a part of cerebral visual impairment (CVI), a class of disorders of the projection and/or interpretation of visual input in the brain.47,105,112 Up to two-thirds of the cases of CVI are thought to be caused by perinatal damage, often the result of preterm birth, but genetic defects might play a significant role in the aetiology of the remaining cases.51,79,119 The majority of persons with CVI exhibit additional clinical features, including intellectual disability (ID).51,53,79,119 We collected 56 individuals with CVI, all born after an uneventful pregnancy and birth, without any identifiable causes, and performed clinical, ophthalmological, and copy number variant investigations (Table S1). This study was approved by the Ethics Committee of the Radboud university medical center (Commissie Mensgebonden Onderzoek, regio Arnhem- Nijmegen), and written informed consent was obtained for all enrolled subjects. For 12 in- Chapter 3.3 dividuals and their parents we performed exome sequencing, by using a trio approach.179,182 Exome sequencing was done in ten of these trios through the Baylor-Hopkins Center for Mendelian Genomics: methods have been reported previously.303 In brief, 1 μg of genomic DNA from each individual was fragmented in a Covaris sonication system. Whole-exome targeted capture was performed in solution with the BCM-HGSC Core design. Sequencing was performed on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA), producing an average of 9-10 Gb of raw data per sample. The sequence reads were mapped and aligned to the Human Genome Reference Assembly GRCh37/hg19 with the BCM-HGSC Mercury pipeline333 at an average depth of coverage of 114x. Variant calling was performed with the Atlas2334 and SAMtools335 algorithms; variant annotation was performed with an intramurally developed annotation pipeline336 based on ANNOVAR337 and custom scripts to incorporate multiple databases to inform on identified variants. In the other two trios we performed exome sequencing on a SOLiD 5500XL platform (Life Technologies, Carlsbad, CA, USA) with the Agilent SureSelect All Exon V4 reagent for target enrichment (Agilent Technologies, Inc.,

191 Chapter 3.3

Santa Clara, CA, USA). The 50 bp paired-end reads were mapped to USCS Genome Browser GRCh37/hg19 Human Genome Reference Assembly, and variants and indels were called with Lifescope v2.1 (Life Technologies, Carlsbad, CA, USA). There was an average coverage of 76x. After quality filtering (variant reads >15%) and exclusion of local and global variants (<1% occurrence) a de novo analysis was performed for all trios. Synonymous variants with no effect on splicing were excluded. In two persons (individuals 1 and 2) de novo missense mutations predicted to be pathogenic were identified in nuclear receptor subfamily 2, group F, member 1 gene, NR2F1 (chr5: 92,921,073 G>C; c.344G>C; p.Arg115Pro and chr5: 92,921,068 C>A; c.339C>A; p.Ser113Arg, respectively; Figure 1, Table S2) [MIM 132890, RefSeq accession number NM_005654.4]. Both were situated in the DNA-binding domain. In addition to CVI, individual 1 had small optic discs with large excavations and ID; individual 2 manifested pale optic discs, small optic nerves on MRI, and developmental delay (Table 1 and Table S3). We screened the remaining 44 CVI persons for mutations in NR2F1 by Sanger sequencing, and identified a third case (individual 3) with a de novo missense mutation, predicted to be pathogenic, in the ligand- binding domain, chr5: 92,923,914 T>C; c.755T>C; p.Leu252Pro. She had excavated, pale optic discs and ID. The Poisson probability of identifying three de novo mutations by sequencing a cohort of 56 persons was calculated for NR2F1. Based on a de novo mutation rate of 1.18x10−8 per position,344 and accounting for GC-content and gene size345-347 the Poisson probability is p=1.40x10-10, indicating that this is highly unlikely to have occurred by chance. This is further strengthened by the consistent clinical phenotype observed in the affected individuals. Point mutations in NR2F1 have not yet been reported, but large deletions spanning NR2F1 and other genes have been associated recently with optic atrophy, visual motor integration deficit, visual perceptual disorder and ID.348 Therefore, we searched for deletions of NR2F1 in both the CVI cohort and the intramural database of the Radboud university medical center, Nijmegen, Netherlands, that includes more than 10,000 array analyses from persons with intellectual disability and/or multiple congenital abnormalities. We identified a 5q15 deletion, arr 5q15(92,845,157-93,679,748)x1 (Human Genome Reference Assembly GRCh37/hg19) comprising NR2F1 with the CytoScan HD array (Affymetrix, Santa Clara, CA, USA) in individual 4, part of the CVI cohort, who had pale optic discs and mild ID (Figure 1). The inheritance of the deletion is unknown, because the deletion was on the paternal allele, but the reportedly phenotypically normal father was not available for testing. We also identified a de novo deletion on chr5:91,064,110-93,896,378 (Human Genome Reference Assembly GRCh37/hg19), arr SNP 5q14.3q15(SNP_A-1810903->SNP_A-1788922)x1 in the intramural cohort (individual 5) with the 250k SNP array (Affymetrix, Santa Clara, CA, USA). Individual 5 had developmental delay and ophthalmological examination showed poor visual acuity, CVI, in the right eye a small pale optic disc, and in the left eye a pale optic disc with a large excavation.

192 NR2F1 mutations cause optic atrophy with ID

A Individual 1 Individual 2 Individual 3 Individual 6

Father

Mother C A C A G T C C G C A G G A G G A G C G T C C T G C T A C G C A A G A G G A G C Val Arg Arg Arg Ser Val Leu Leu Arg Lys Arg Ser c.344G>C c.339C>A c.755T>C c.335G>A p.Arg115Pro p.Ser113Arg p.Leu252Pro p.Arg112Lys

B p.Ser113Arg p.Arg112Lys p.Arg115Pro p.Leu252Pro

N DBD LBD C 0 83 154 221 381 423

C Chromosome 5

hg19 91,0 91,5 92,0 92,5 93,0 93,5 Mb

5q14.3 5q15 Chapter 3.3

Individual 5 AK056485 Individual 4 NR2F1 KIAA0825 FAM172A POU5F2

figure 1: Phenotype and genotype of the individuals with optic atrophy (A) Confi rmation by Sanger sequencing of thede novo mutations in NR2F1 [NM_005654.4] found by exome sequencing (individuals 1, 2 and 6), the de novo mutation in NR2F1 found by cohort screening (individual 3), and segregation analyses. The pictures of the individuals show nonspecifi c facial dysmorphisms. (B) Schematic representation of NR2F1, containing a DNA-binding domain (DBD) and a ligand-binding domain (LBD). The positions of the mutations identifi ed in the aff ected individuals are indicated. (C) Overview of the 5q14-q15 region encompassing the two overlapping microdeletions of individual 4 (lower red bar) and 5 (upper red bar) comprising NR2F1. The inheritance of the deletion in individual 4 was unknown, because the father was not available for testing and the deletion was on the paternal allele. The deletion of individual 5 was de novo. The pictures of the aff ected individuals show nonspecifi c facial dysmorphisms.

193 Chapter 3.3

Table 1: Genotype and phenotype of the affected individuals Individual 1 Individual 2 Individual 3 Individual 4 Individual 5 Individual 6 Defect in NR2F1 c.344G>C c.339C>A c.755T>C Deletion Deletion c.335G>A p.Arg115Pro p.Ser113Arg p.Leu252Pro (0.83 Mb) (2.85 Mb) p.Arg112Lys Gender Male Female Female Female Female Female Age 12y 2+4y 18y 24y 4y 35 y Development IQ 48 DD IQ 55-65 IQ 61-74 DD IQ 52 Behavioural - - - - - OCD, autism abnormalities OFC (centile) 98th 55th 55th 99th 50th 13th CVI + + + + + - Optic disc Small and large Pale Pale and large Partial pale Small, pale and Pale abnormalities excavation excavation large excavation MRI brain Normal Small optic ND ND Normal Normal nerve and chiasm DD = developmental delay, ND = not done, OCD = obsessive-compulsive disorder, OFC=occipital frontal head circumference.

In parallel, a single additional individual with congenital optic atrophy and ID was enrolled through the Baylor-Hopkins Center for Mendelian Genomics study (individual 6, BAB3468, Figure 1 and Table 1). This 35-year-old female also had autism spectrum disorder with marked obsessive-compulsive behaviours. The pregnancy and delivery had been uneventful (Table S1). Family history was negative for optic nerve atrophy, ID, developmental delay, and autism. The family was consented through a research protocol approved by the Institutional Review Board (IRB) of Baylor College of Medicine. DNA samples from the proband and her unaffected parents were submitted for exome sequencing by using a trio approach as described above at an average depth of coverage of 107x and with >91% of the target bases covered at a minimum of 20x. Inheritance of the identified variants was examined with the DeNovoCheck algorithm.182 Analysis of the variants identified in the proband under a sporadicde novo model of inheritance revealed a missense mutation (chr5: 92,921,064 G>A; c.335G>A; p.Arg112Lys) in NR2F1. This variant was situated in the DNA-binding domain and was predicted to be pathogenic. As individual 6 manifested optic nerve atrophy without CVI a cohort of mainly nonsyndromic persons (n=37) with optic nerve abnormalities was screened for NR2F1 variants by Sanger sequencing (Table S4). In a 68-year-old male with optic atrophy a missense mutation was identified, chr5:92,924,068 G>C; c.909G>C; p.Gln303His. However, this variant was also present in his unaffected sister and son and was interpreted as a nonpathogenic variant. NR2F1 encodes a conserved orphan nuclear receptor protein (nuclear receptor subfamily 2, group F, member 1), that, activated by a yet unknown ligand, regulates transcription.349 It belongs to the nuclear receptor superfamily, of which several members already have been impli-

194 NR2F1 mutations cause optic atrophy with ID cated in human disease: NR2E3 [MIM 604485] in retinitis pigmentosa,350 ESRRB [MIM 602167] in deafness,351 NR0B1 [MIM 300473] in congenital adrenal hypoplasia,352 and NR2F2 [MIM 107773] in congenital diaphragmatic hernia.353 NR2F1 has a classic nuclear receptor structure with two main domains, a functional DNA-binding domain (DBD) formed by two zinc-finger structural domains and a ligand-binding domain (LBD) with two highly conserved sequence regions.354 Interestingly, three of the four point mutations that we identified localize to the DBD of NR2F1, probably affecting the binding of NR2F1 to its transcriptional targets. To investigate the functional effects of our identified point mutations in NR2F1, we established a reporter assay. In this assay luciferase is under control of an NR2F1 activated promoter and cells are cotransfected with this plasmid and either wild-type (WT) or mutant Nr2f1 constructs. The luciferase activity reflects the transcriptional activity of the constructs. The expression plasmids of the mouse Nr2f1 - COUP-TFI, and the NGFI-A (−168/+33) promoter luciferase reporter in pXP2 were previously described.355,356 All point mutations were generated with the QuickChange site-directed mutagenesis kit (Agilent). For luciferase assays, HEK293T cells were plated in 24-well plates (1x105 cells per well) 24 hr prior to transfection. Cells were transfected with 5 ng of reporter per well with Lipofectamine 2000 following the manufacturer’s instructions. Forty-eight hr after transfection, cells were harvested and luciferase activity was measured by the luciferase assay system (Promega). As indicated in Figure 2, all DNA-binding y i t i v t a c A ) - e I G F e r a s N i f - L u c e ( p X P 2 i v Chapter 3.3 e l a t R T s o r y t W e c 1 5 P r o 1 2 L V 1 3 A r g S e r 1 A r g 1 A r g 1 L e u 2 5 P r o p . p . p . p .

WB: Flag

WB: β-actin

Figure 2: In vitro luciferase reporter assay for NR2F1 point mutations Effects of the Nr2f1 (COUP-TFI) mutations on activation of an NGFI-A promoter-driven reporter. Top shows that pXP2-NGFI-A was cotransfected with 5 ng FLAG-tagged Nr2f1 (COUP-TFI) WT, FLAG-tagged Nr2f1 (COUP-TFI) mutations or empty vector into HEK293T cells as indicated. The cell sample transfected with pXP2-NGFI-A and vector served as the control, and its value (relative luciferase activity) was set as 1. The relative luciferase activities of the other samples are normalized to this control. Data are means ± SD (n = 3). Bottom shows immunoblot analysis of Nr2f1 (COUP-TFI) expression in transfection. Results indicate similar expression of WT and mutant Nr2f1 (COUP-TFI) constructs.

195 Chapter 3.3 domain mutants (p.Arg112Lys, p.Ser113Arg, and p.Arg115Pro) and the ligand-binding domain mutant (p.Leu252Pro) lost their ability to fully activate NR2F1 - COUP-TFI reporter. This result supports our contention that persons who have these mutations do not have fully functional NR2F1 - COUP-TFI product. Additionally, the function of NR2F1 has been investigated in mice. The mouse ortholog, Nr2f1, is expressed in various tissues throughout development, with important functions in cell fate determination, differentiation, migration, and survival, and an apparently important role in organogenesis354. In the developing nervous system, it is mainly expressed at high levels in the optic nerve, thalamus, and pallium (future cortex).357-359 Studies of Nr2f1 knockout mice indicate that the protein is important for neurodevelopment, including oligodendrocyte differentiation (partly in vitro experiment),357 cortical patterning,360-362 and guidance of thalamocortical axons,363 as well as eye and optic nerve development.356,364 Furthermore, overex- pression of Nr2f1 in mouse neuronal differentiating teratocarcinoma PCC7 cells antagonized neuronal differentiation induced by retinoic acid signalling.365 Given our results from the in vitro reporter assays and because deletions of NR2F1 give rise to a similar phenotype, we hypothesize that the heterozygous mutations identified in our affected individuals cause loss of function and that the observed phenotypes are due to haploinsufficiency. Interestingly, it appears that the latter are restricted to brain and optic nerves, because no other major organ abnormalities are observed in our cohort of patients with either NR2F1 point mutations or deletions. This might be due directly to levels of NR2F1 reduced below a threshold necessary during brain development or indirectly via the target genes it regulates for specific expression in brain development. Brown et al. proposed haploinsufficiency of NR2F1 as the cause of deafness and multiple congenital anomalies in an individual with NR2F1 deletion and paracentric inversion inv(5)(q15q33.2).366 The absence of deafness and multiple congenital anomalies in the affected individuals 4 and 5 suggests that these might be caused by the more complex chromosomal rearrangement identified in the reported person, although phenotypic variability cannot be ruled out at this time. In addition to the optic nerve abnormalities, five out of six individuals were diagnosed with CVI. This potentially could reflect thalamocortical axon projection disorders, as reported in Nr2f1 knockout mice.363 Until now, optic atrophy in combination with optic disc excavation was thought to be the result of perinatal damage.80,81,102 We showed, however, that optic disc excavations can also be present in individuals with a genetic cause of CVI (individuals 1, 3 and 5). NR2F1 investigation should, therefore, also be considered in preterm children with CVI. In conclusion, four individuals with optic atrophy and CVI of unknown cause had heterozygous de novo point mutations in NR2F1, and two persons with a similar phenotype had deletions comprising NR2F1. Our findings indicate that NR2F1 has an important role in the development of the visual system and that haploinsuffiency can lead to optic atrophy with intellectual impairment.

196 NR2F1 mutations cause optic atrophy with ID

Supplemental tables

Table S1: Description of CVI cohort Person In Age at Gender Gestational Intellectual NGS (platform) manuscript examination in age (weeks) disability/ years (+ months developmental when under 4 delay years) CVI1 Individual 1 12 M 42 + + (Illumina Hiseq 2000) CVI2 3+8 M 42 + + (Illumina Hiseq 2000) CVI3 1+7 M At term + + (Illumina Hiseq 2000) CVI4 21 M 40 + + (Illumina Hiseq 2000) CVI5 12 M 37 + + (Illumina Hiseq 2000) CVI6 11 F 42 + + (Illumina Hiseq 2000) CVI7 8 M 40 + + (Illumina Hiseq 2000) CVI8 7 M 38 + + (Illumina Hiseq 2000) CVI9 7 F 41 + + (Illumina Hiseq 2000) CVI10 7 M 38 + + (Illumina Hiseq 2000) CVI11 Individual 2 4 F 41 + + (SOLiD 5500XL) CVI12 4 F 40 + + (SOLiD 5500XL) CVI13 9 M 40 + CVI14 Individual 3 18 F At term + CVI15 Individual 4 24 F 36 + CVI16 3+7 M 37 + CVI17 3+4 M 35 + CVI18 2+10 M 41 + CVI19 2+1 F 40 +

CVI20 1+6 M 37 + Chapter 3.3 CVI21 38 F At term + CVI22 36 M 42 + CVI23 33 F At term - CVI24 33 M 41 + CVI25 30 F 40 + CVI26 29 M At term + CVI27 28 M At term + CVI28 28 F At term + CVI29 26 F At term + CVI30 26 M 39 + CVI31 23 F 39 + CVI32 22 F 42 + CVI33 20 F 40 + CVI34 18 M At term +

197 Chapter 3.3

Table S1: Description of CVI cohort (continued) Person In Age at Gender Gestational Intellectual NGS (platform) manuscript examination in age (weeks) disability/ years (+ months developmental when under 4 delay years) CVI35 17 M 40 + CVI36 17 M At term - CVI37 15 F 40 + CVI38 15 M 42 + CVI39 2 F 38 + CVI40 14 M 32 - CVI41 13 M 40 - CVI42 13 F At term + CVI43 13 F 40 + CVI44 12 M 42 + CVI45 10 M 39 + CVI46 9 M 40 + CVI47 7 M 41 + CVI48 7 F 40 + CVI49 7 M 38 + CVI50 6 M 41 + CVI51 6 M 37 + CVI52 6 M 37 + CVI53 6 F At term + CVI54 5 M At term + CVI55 5 M 39 +

Table S2: Pathogenicity prediction of the identified mutations in NR2F1 Individual 1 Individual 2 Individual 3 Individual 6 Coding DNA change c.344G>C c.339C>A c.755T>C c.335G>A Protein change p.Arg115Pro p.Ser113Arg p.Leu252Pro p.Arg112Lys PhyloP 5.05 1.90 4.56 5.05 Conservation down to C. elegans C. elegans C. elegans C. elegans Pholyphen2 (score) Probably damaging Probably damaging Probably damaging Probably damaging (1.00) (1.00) (0.99) (0.995) Sift (score) Deleterious (0.00) Deleterious (0.00) Deleterious (0.00) Deleterious (0.00) Mutation Taster (Score) Disease causing (0.99) Disease causing (0.99) Disease causing (0.99) Disease causing (0.99) MutPred 0.888 0.848 0.939 0.947 EVS frequency 0 0 0 0 dbSNP frequency 0 0 0 0

198

Chapter 3.3

Table S3: Additional phenotype of the individuals in which a NR2F1 defect was identified Individual 1 Individual 2 Individual 3 Individual 4 Individual 5 Individual 6 Gender M F F F F F Birth weight (grams) 3510 (p7) 3855 (p70) 3780 (p70) 2500 (p50) 4150 (p86) 2800 (p12) GA (weeks) 42 41 AT 36 41 40 Age at investigations (years) 12 2+4 18 24 4 35 Height (cm) 159 (p84) 87 (p16) 185 (p99) 170 (p50) 107 (p55) 158 (p21) Dysmorphisms Protruding ears, long nostrils, Epicanthal folds, upturned nose Upslanting palpebral Tapering fingers, extra flexion crease dig Broad high nasal bridge, prominent Right helix incompletely folded. Prominent prominent alea nasi, flattened point, broad mouth, full lips, fissures, small nasal ridge, IV right, II-III syndactyly toes. antihelix of the ear, clinodactyly dig V. Darwin’s tubercle on right ear. Pointed chin. thorax, long fingers and toes. simple protruding ears with retrognathia, high palate, Synophrys (familial). upturned ear lobes, tapering long fingers and toes. fingers and fetal fingers pads, curly slow growing hair. Hearing ------Other abnormalities Hypotonia. Hypotonia. Neonatal feeding problems. Neonatal feeding problems. Mild slowing in rapid alternating movements. Mild spasticity. Refraction error OD S+7.25=C-2.25x164” OD S+3 OD S+0.25=C-0.50x4” OD S 0=C-1.00x111” OD S+1.75 OD S-2.00=C+4.00x180” OS S+6.75=C-1.75x30” OS S+3 OS S-0.25=C-0.75x11” OS S-0.71=C-0.25x50” OS S+1.75 OS S-2.00=C+4.00x180” Correction of refraction + - - + - - Visual acuity OD 0.06 ND 0.08 0.08 ND 0.1 Visual acuity OS 0.03 ND 0.1 0.2 ND 0.1 Visual acuity ODS 0.125 20/260 (0.08) 0.16 0.25 0,2 0.1 Method (distance) LH (3 m) TAC (55 cm) Landolt C 2.6’ (5 m) Landolt C 2.6’ (5 m) TAC (55 cm) Snellen Strabismus + + + + + + Oculomotor disturbance - - + - + - Nystagmus Latent. - Latent. Latent. Latent. - Visual field defects + + + - + ND Slit lamp examination Iris transillumination. - - Deep anterior chamber, mild lens Slight iris transillumination Bilateral keratoconus. opacity. Optic disc Small and large excavation. Pale. Pale and large Partial pale. OD small and pale. Pale. excavation. OS pale and large excavation. Macula Not recognizable. Normal. Normal. Normal. Normal. Inner retinal atrophy with muted umbo and foveal light reflexes. VEP (age) Flash: late response; pattern onset: Flash: long latency times (10 Flash:normal; pattern ND ND ND late response; pattern reversal: months). reversal: long latency (8 normal respons (8 years). years). ERG (age) Normal (8 years). ND Normal ( 8 years). Normal (11 years). ND ND Other ocular features Crowding, fluctuating visual Crowding. Keratitis sicca managed with punctal plugs performance, excentric fixation. OU. GA = gestational age, ND = not done.

200 NR2F1 mutations cause optic atrophy with ID

opacity. Chapter 3.3 Optic disc Small and large excavation. Pale. Pale and large Partial pale. OD small and pale. Pale. excavation. OS pale and large excavation. Macula Not recognizable. Normal. Normal. Normal. Normal. Inner retinal atrophy with muted umbo and foveal light reflexes. VEP (age) Flash: late response; pattern onset: Flash: long latency times (10 Flash:normal; pattern ND ND ND late response; pattern reversal: months). reversal: long latency (8 normal respons (8 years). years). ERG (age) Normal (8 years). ND Normal ( 8 years). Normal (11 years). ND ND Other ocular features Crowding, fluctuating visual Crowding. Keratitis sicca managed with punctal plugs performance, excentric fixation. OU. GA = gestational age, ND = not done.

201 Chapter 3.3

Table S4: Optic nerve abnormality cohort Person Variant identified Gender Age (years) Disease ONA1 c.909G>C; p.Gln303His M 68 OPA ONA2 V 68 OPA ONA3 M 60 OPA ONA4 V 39 OPA ONA5 M 48 OPA ONA6 M 46 OPA ONA7 M 41 OPA ONA7 M 74 LHON ONA8 M 69 LHON ONA9 M 64 LHON ONA10 V 63 LHON ONA11 M 63 LHON ONA12 M 58 LHON ONA13 M 57 LHON ONA14 V 56 LHON ONA15 V 56 LHON ONA16 M 53 LHON ONA17 M 52 LHON ONA18 M 52 LHON ONA19 V 51 LHON ONA20 M 51 LHON ONA21 V 47 LHON ONA22 M 45 LHON ONA23 M 44 LHON ONA24 V 38 LHON ONA25 M 34 LHON ONA26 M 33 LHON ONA27 V 25 LHON ONA28 M 25 LHON ONA29 V 21 LHON ONA30 M 13 LHON ONA31 M 13 LHON ONA32 V 9 LHON ONA33 M 50 LHON ONA34 M 3 Opticopathy and developmental delay ONA35 V 33 Optic nerve abnormaliy ONA36 V 30 OPA and deafness

OPA = optic atrophy, LHON = Leber hereditary optic neuropathy.

202

Chapter 4

Overall results of the CVI study

4.1 Characteristics and results of the cohort

To investigate the genetics of cerebral visual impairment (CVI) a cohort of 828 patients with low vision and no major ocular disorders was obtained. After applying stringent criteria, described in chapter 1.5, 56 patients were included for further dysmorphological and ophthalmological investigations (Figure 1). A thorough clinical examination was performed, and the clinical characteristics are presented in Table 1. Subsequently DNA investigations, including targeted gene analysis, microarray and whole exome sequencing (WES) were performed. The identified aberrations were classified into known CVI-associated aberrations (CVI had been previously reported for this specific region or gene), candidate aberrations for CVI (CVI had not been previously reported for this specific region or gene), and unlikely to be related to CVI (based on variant and gene characteristics). In 26 patients a microarray had not been performed previously, thus a genome wide CytoScan HD array (Affymetrix Inc., Santa Clara, California, USA) was done. This led to the identification of five deletions, classified into known CVI-associated deletions (n=2) and candidate deletions for CVI (n=3) (Table 2). In 32 patients whole exome sequencing (WES) was accomplished, and a known CVI-associated aberrant gene was identified in 9 individuals, and a candidate aberrant gene for CVI was found in 16 individuals.

Initial cohort (n=828) - Risk factor and/or known genetic disorder (n=631) - Deceased (n=58) Eligible (n=139)

- Declined (n=53) - Unreachable (n=30)

Included for study described Chapter 4 in chapter 3 and 4 (n=56)

Figure 1: Flow diagram of the patient inclusion for the study described in chapter 3

207 Chapter 4

Table 1: Cohort characteristics (n=56) Characteristic Number of patients Gender Male 35 Female 21 Age group <4 years 8 4-10 years 19 11-20 years 13 >20 years 16 Visual acuity ≥0.05 54 <0.05 2 Fixation abnormalities Yes 17 No 39 Visual field defects Yes 27 No 26 Unknown 3 Developmental delay and/or intellectual disability Yes 52 No 4 Head circumference Microcephaly (<3th centile) 12 Normocephaly (3th-97th centile) 38 Macrocephaly (>97th centile) 5 Unknown 1 Abnormality on brain MRI Yes 28 No 15 Not assessed 13 Epilepsy Yes 15 No 41 Major congenital abnormality (not the brain) Yes 10 No 46 Facial dysmorphisms >2 Yes 49 No 7 Hearing loss Yes 3 No 52 Unknown 1

208 Overall results of the CVI study

Table 2: Identified aberrant regions or genes (possibly) associated with CVI Patient ID WES Known CVI-associated aberration Candidate aberration for CVI Reference 1 + NR2F1 191 2 + ARGHEF10L, GABRB2 367 3 + 4 + UHMK1 367 5 + AHDC1 367 6 + NR2F1 368 7 + 8 + 9 10 + AMOT 367 11 + DLG4 367 12 + KCNQ3 367 13 + NR2F1 ACP6 191 14 + KAT6B 15 + ATP6V1A, UFSP2 367 16 + 17 + 18 + DCAF6, GRIN1, SLC25A16 367 19 + 20 + PGAP1 257 21 + GRIN2B, ZFP30 367 22 + KCTD19, SOX5 367 23 + 24 + 25 + SHOC2 26 + RERE, SLC1A1 367 27 + SLC35A2 367 28 + NGLY1 367 29 Chapter 4 30 31 32 33 34 +* RARS 369 35 +* RARS 369 36 del 10p12 370 37 del 4q28.3q31.1 251 38 39

209 Chapter 4

Table 2: Identified aberrant regions or genes (possibly) associated with CVI (continued) Patient ID WES Known CVI-associated aberration Candidate aberration for CVI Reference 40 del 5q15 191 41 42 43 44 del 1p36.33p36.23 45 46 +* MEF2C 47 +* MAGEL2 48 +* PLP1 371 49 del Xp22.11 50 51 NR2F1 191 52 53 54 55 56 Number of genetic 11 (20%) 19† (34%) diagnosis *WES was initiated by others and led to a diagnosis. †In patient 13 also a variant in a known CVI-associated gene was identified. Therefore the candidate gene is not counted in the total number of diagnosis. Patients 8 and 9, and patients 34 and 35 were sibs.

210 Overall results of the CVI study

4.2 Conclusions and implications

In this thesis we studied files followed by a study to identify the molecular diagnosis underlying low vision CVI. Of the initial cohort of 828 individuals 58 patients (6.8%) were deceased. This is a high rate compared to the overall mortality rate in the Netherlands, i.e. 0.8% in 2013 (all ages) (http:// statline.cbs.nl/, accessed on June 2015). Taken into account that the patients with CVI were mainly children, this indicates that CVI can be present in seriously ill and vulnerable patients. From the 828 patients, 56 were included for the study in chapter 3 and 4. This 56 individuals were selected in order to exclude other potential (genetic) causes. We identified 11 known CVI-associated aberrations, establishing a diagnosis in 20% of the individuals. Five of them (9%) had an aberration in NR2F1 leading to Bosch-Boonstra-Schaaf optic atrophy syndrome. Although it is a small and selected cohort, NR2F1 aberrations are very likely to be a frequent cause of CVI. For comparison, the most common cause of ID is Down syndrome, which accounts for about 8% of the individuals with ID.372 Screening the whole initial cohort of 828 CVI-patients on NR2F1 aberrations would be interesting in order to determine the prevalence of NR2F1 more accurately. There might be patients with Bosch-Boonstra-Schaaf optic atrophy syndrome in whom CVI had been addressed to perinatal problems. In addition, we excluded patients with West syndrome for the study in described in chapter 3, but West syndrome is now also associated with Bosch-Boonstra-Schaaf optic atrophy syndrome.373 Finally, in 19 patients (34%) a candidate genetic aberration for CVI was obtained, and recurrence is warranted to establish the relation with CVI, making a possible yield of ~50%. Chapter 4

211

Chapter 5

General discussion and future perspective

5.1 Challenges in variant interpretation

There are considerable challenges in the assessment of the pathogenicity of single nucleotide and copy number variants in human genomes. The use of prediction programs, variant databases, segregation, phenotypic analyses, functional assays, and animal models can help in this interpretation. However, one cannot always come to a clear conclusion, leaving the patient and parents with uncertainties. Below, the interpretation of single nucleotide variants identified by whole exome sequencing (WES) in patients with cerebral visual impairment (CVI) will be discussed.

Variant characteristics and gene function The presence of a variant in healthy controls is an important reason to classify a variant as nonpathogenic. However, how healthy is a healthy control? One of the most recent and largest databases (~60,000 individuals; http://exac.broadinstitute.org/) for healthy controls also includes individuals with schizophrenia and bipolar disorders. Moreover, there are disorders with a milder phenotype and/or a wide clinical spectrum. For example, the range of IQ of the individuals with a NR2F1 mutation, a recently identified gene for CVI, is between 48 and normal (chapter 3.3 and www.nr2f1gene.com). Based on the frequency of the investigated disorder, the severity of the disorder, and the inheritance pattern, the allele frequency can be estimated and used to define a frequency threshold, as was done in chapter 3.1 for CVI. In addition, it is important to investigate whether the variant segregates with the phenotype. In chapter 3.1 we could exclude two thirds of the predicted pathogenic X-linked variants based on segregation analyses in nonaffected second degree relatives. In addition to databases with control individuals there are databases in which variants in disease genes are collected and assessed on pathogenicity, for example the HGMD or Leiden Open Variation Databases (LOVDs).374,375 When these databases and segregation analyses are not helpful, the variant can be judged on several aspects as pointed out in chapter 3.1. The conservation of the wild-type nucleotide and amino acid can be assessed, and it can be predicted whether the variant leads to an aberrantly functioning protein. There are many prediction programs, and the application of two or more programs yields more reliable results.376 We used SNPs&Go, Mutpred and Polyphen-2, because they asses different aspects of the variant and have a relatively high sensitivity and specificity according to the study of Thusberg et al.377 Other commonly used tools, which are also applicable for a large dataset, are SIFT, Condel and the more recently introduced program Combined Annotation Depen- Chapter 5 dent Depletion (CADD),378-380 but there is no consensus which tools should be used when assessing missense variants. In addition, the gene function, and its possible involvement in neurological disorders, can be predicted based on gene ontology and mouse phenotype terms (http://www.informatics. jax.org/).381,382 Moreover, sometimes a gene is guilty by association: the gene product can

215 Chapter 5 have an interaction with a protein encoded by a gene known to be associated with the same or a similar disease (RERE binds NR2F1), belongs to the same protein family (DLG4 belongs to the same protein family as DLG3), or is involved in a pathway (PGAP1 in GPI-anchoring pathway) that is associated with a disease. All these aspects can be helpful in the interpretation of a variant, but there remain always false-positives and false-negatives. For example the method we used to interpret the variants was based on two previously published papers on WES and whole genome sequencing (WGS) in patients with intellectual disability (ID).182,256 In four out of 100 patients the assignment of an identified variant to be causative turned out to be false-positive. However, more than two years after the publication of the first paper on 100 patients with ID already more than half of the proposed candidate genes have been confirmed through various methods to be ID-associated genes, supporting the validity of this method.182

Recurrence and phenotyping An important aspect in assessing the pathogenicity is to compare the phenotype of individuals with an aberration in the same gene. In the patient in which we identified NGLY1 mutations (chapter 3.1), the phenotype showed strong similarity with the previously reported patients, including the rare feature of ‘absence of tears’.259,261 In contrast, when a variant is identified in for example NR2F1, and the patient shows significant facial dysmorphisms and no visual impairment, the causality of the variant remains uncertain. Sometimes however, the phenotype is broader than previously thought, as variants in KCNQ3 illustrated. Mutations in KCNQ3 were associated with benign epilepsy, without CVI or ID, but subsequently, we and others identified variants in patients with CVI and ID.185,285-295 The use of WES not only broadens the spectrum of known disease-associated genes, but also many new associations between genes and phenotypes are being made. However, strong conclusions come from additional patients with a similar phenotype. In chapter 3.3 we calculated that the chance of identifying three patients with de novo NR2F1 variants in the cohort was very small, p=1.40x10-10. Since this publication already more than 20 patients have been identified with an overlapping phenotype including CVI, corroborating the causality of NR2F1 mutations in Bosch-Boonstra-Schaaf optic atrophy syndrome. However, for other candidate genes identified in chapter 3.1 and chapter 4.1 additional data are needed to assess causality. If loss-of-function mutations have been identified, additional cases can be identified by searching for overlapping deletions. For a truncating variant the loss-of-function may be clear, but for missense variants only functional assays can confirm the loss-of-function. When there are overlapping deletions, comparing phenotypes is valu- able. Databases like ECARUCA and DECIPHER contain many deletions with clinical data and it is possible to contact the clinician involved (http://umcecaruca01.extern.umcn.nl:8080/ ecaruca/ecaruca.jsp and https://decipher.sanger.ac.uk/).148,149,383 Other cases with mutations in the same gene can also be found by screening additional patient cohorts. For the NR2F1

216 General discussion and future perspective gene the CVI cohort was screened by Sanger sequencing and this revealed an additional patient with a de novo NR2F1 variant. Cost-effective sequencing methods, such as a method based on molecular inversion probes (MIPs) opens the possibility to screen the identified candidate CVI genes on the entire initial CVI cohort (n = 828).280 Moreover, also other large cohorts can be screened, such as an ID patient cohort, since ID is a common finding in CVI patients and CVI is probably underreported and/or underdiagnosed. Additional cases can also be found through collaborations. In addition to presentation at congresses or publications, web based programs, such as gene matcher or human disease gene websites have been developed (https://genematcher.org/ and http://humandiseasegenes.com/). A lot of genetic data, however, are not published or shared in a public domain. Very likely, this will be overcome thanks to the efforts of the parents and patients. The RERE-facebooksite of a parent initiated the collaboration between another research group and us. Also for other newly identified genes parents played an important role. Through the identification of the NGLY1 associated disorder, parents not only recruited additional patients, they even suggested a functional assay.384,385

Functional investigations Not always is the phenotype distinct enough or are there enough patients identified to assess the pathogenicity of the variant. Another way to obtain additional proof is by functional investigations. Depending on the function of the gene, assays are developed to test whether a specific variant influences its function. For example, we identified missense variants NR2F1in (chapter 3.3), and the transcriptional activity of the mutant construct was decreased in patients with Bosch-Boonstra-Schaaf optic atrophy syndrome. In contrast, the mutant construct based on the non-segregating missense variant in a patient with non-syndromal progressive optic atrophy showed normal transcriptional activity (data not shown). This indicates that an assay can be used to test newly identified missense variants on their pathogenicity for a specific syndrome. However, these types of assays investigate only one single function of a protein. Sometimes a protein is involved in multiple processes and different variants could affect different functions, which might lead to different disorders. Another possibility to investigate the pathogenicity of a variant and to learn more about the function of the gene are animal models, for instance fruit flies, zebrafish, or mice.386-388 There are obvious differences, and not all genes are conserved between species.387 In addition, similar genetic defects can result in different or no phenotype at all. For instance, no significant photoreceptor disease is present in Myo7a null mice, whereas in human MYO7A deficiency Chapter 5 leads to Usher syndrome type 1B.389,390 Nevertheless, when an orthologue is present and the phenotype of the genetically modified animal is reminiscent of the human disorder, animal models are important to investigate the pathophysiology and to test potential therapeutic interventions.391 Moreover, tools have been developed to generate ‘knock out’ and ’knock in’ animals, in which the mutated gene can be conditionally expressed at a specific life stage or

217 Chapter 5 in a specific tissue.388 Subsequently, many phenotypic aspects can be investigated, including anatomy and behavioural features, which can reflect cognitive function.386,387,391 It is possible to assess vision in for example fruit flies, zebrafish and mice, making these animal models suitable to investigate CVI.392-394

5.2 CVI: a separate entity?

More than 90% of the patients in this study were intellectually disabled (chapter 2.1 and chapter 4.1), and several of the genes we identified have been implicated in ID. This might raise the question whether CVI is an inherent feature of ID. CVI with a visual acuity of ≤0.1 is indeed much more prevalent in persons with cognitive and/or motor impairment compared to the prevalence in persons without ID, 60% vs 6% respectively.55 Vice versa, in individuals with ID CVI with low vision (visual acuity ≤0.3) is present in 5%-20% of the cases.225,395,396 In addition, it has been shown that in the more severe ID cases, the number of individuals with low vision increases (Table 1).225,397-399 This increase in prevalence of low vision is not due to uncorrected refraction errors, thus the reason remains to be elucidated.397

Table 1: Visual acuity in persons with ID Level of ID* Developmental level* VA > 0.5 VA ≤ 0.3† Mild ID (IQ 50-70) 9-12 years ~90%225,397 ~3-25%225,397-399 Moderate (IQ 35-49) 6-9 years ~75%397 Severe (IQ 20-34) 3-6 years ~45%397 ~40-75%397-399 Profound (IQ <20) < 3 years ~25%397 *based on ICD-10 (http://apps.who.int/classifications/icd10/browse/2015/en). The individuals with ID are children and adults. †In the general Dutch population, age between 15 - 49 years, the prevalence of low vision is 0.3%.400 VA = visual acuity.

It would be interesting to plot the visual performance in patients with CVI, such as visual acuity, crowding and visual fields, against the developmental age. In our study this was not possible, because at the moment of the ophthalmological examination the developmental age was not recently measured. Nevertheless, an inverse relation between the cognitive level and the visual performance can be expected. On the other hand, there is evidence that discrepancy in developmental level between various neurological functions (such as cognition and speech) can occur. For instance, in Wil- liams syndrome some aspects of speech are relatively well preserved, whereas in Angelman syndrome the development of speech is relatively more affected compared to their cognitive development.401,402 Moreover, FOXP2 aberrations lead to a severe impairment of speech in patients, while their cognitive development is hardly affected.403,404 For CVI, the same might

218 General discussion and future perspective apply: in some disorders the visual system functions relatively well, and in others relatively bad. When there is localized brain damage, e.g. ischaemia of the visual cortex, the explanation for this discrepancy is obvious. However, for more global (and possible genetically induced) brain malfunctioning, it is more challenging to unravel the underlying mechanism of the discrepancy in the degree of impairment between neurological functions.

5.3 An outlook

Diagnosing CVI As discussed in the introduction there is no consensus about the diagnostic criteria for CVI. Moreover, in clinical practice the differential diagnosis of primary optic atrophy and delayed visual maturation (DVM) can be difficult. There are measurable features of CVI, such as slow vision (saccade latency), crowding and visual fields defects, which have been investigated.42,60,72,76 In clinical practice, these assessments can provide additional support for the diagnosis of CVI. Besides the current practice of comprehensive ophthalmological, neurological, and neuropsychological investigations, additional investigations such as genetic investigation and functional MRI (fMRI) are becoming helpful in the diagnosis of CVI.

Therapy in CVI Stimulation, guidance and support are essential for CVI patients,52,405 but only limited research have been performed to measure the effect of stimulation programs in CVI.42,193 Especially in young patients with acquired brain damage, it was shown that spontaneous recovery occurred and the visual processing was reorganised towards undamaged areas.406 In addition, in adults with a hemianopia due to stroke part of the visual field could be regained by visual field training.407-409 However, it is imaginable that a genetic disorder that affects the whole brain needs an approach, different from localized damage to the visual system. By knowing the aetiology it can be examined whether there is a specific ‘CVI-profile’ for different conditions. In chapter 2.1 we showed that there is a difference in ocular features between CVI due to an acquired cause compared to CVI in patients with a genetic disorder. In line with this, it is likely that there are differences in CVI features between different genetic disorders. For instance, that in some genetic disorders the fixation and visual fields are mainly affected, whereas in other genetic disorders crowding is a major feature. Subsequently, it can be investigated which stimulation programs can be best used for specific aetiologies and ‘CVI-profiles’. More- Chapter 5 over, as discussed in chapter 1.2, there is a critical period for the development of the visual system so the timing of the intervention is likely important.

219 Chapter 5

Unravelling the pathophysiology Understanding the pathophysiology will help answering the raised questions and cope with challenges: is the variant in this specific gene causal, why is the visual system sometimes affected more severely, which part of the visual system is affected, what is the pathophysi- ological difference between DVM and CVI, is there transsynaptic retrograde degeneration (secondary optic atrophy) or an endogenous defect of the optic nerve (primary optic atrophy), and from which therapy might the patient benefit most? The use of animal models and fMRI are different approaches for further investigating the disturbed development of the visual system and this is illustrated below. Haploinsuffiency of NR2F1 leads to optic nerve abnormalities, CVI and ID (chapter 3.3). In mouse studies it was shown that the subplate neurons differentiation was compromised and that the thalamocortical axon projections were disturbed during visual development, which might lead to an impairment of the transport of visual stimuli towards the cortex.363 It may be possible that other thalamocortical projections, e.g. hearing, would also be disturbed,410 but hearing loss is only present in a minority of cases.348,368 To investigate the effect of the disturbed thalamocortical projections on vision, conditional knock-out mice with tissue-specific deficiency of NR2F1 in, for example the thalamus or subplate, could be developed.388 This could help in the understanding of the origin of the visual deficits inNR2F1 patients. Another approach to gain insight would be fMRI, that has been used to visualize the abnormal visual processing in individuals with acquired localized damage.406 In individuals with Neurofibromatosis Type 1 with higher visuospatial impairment (a higher visual deficit), deficient activation was seen in several visual areas.411 This deficient activation was not correlated with IQ. However, for many young children and patients with ID fMRI can be too demanding, because of the noise and the necessity to remain without motion for about 4-8 minutes.412 Therefore, fMRI studies in sedated and natural sleeping children exposed to flashing lights have been performed, which results in activation of the visual cortex.412-418 In one report the flash induced activated brain volume increased with the increasing severity of the occipital periventricular leukomalacia (which is strongly correlated with CVI), but visual function was not investigated in this group.413 In contrast, in children with established visual function deficits due to perinatal damage, the area of activation was smaller.414,418 Further research is necessary to unravel whether different aetiologies of CVI lead to different patterns of activated brain areas. In addition, this fMRI pattern might correlate with the presence or absence of CVI-features (e.g. low visual acuity, visual field defects, prosopagnosia). Moreover, it can be investigated whether fMRI patterns are of predictive value for the visual outcome. For this research homogeneous study groups are required, which can be obtained by select- ing patients with a similar gene defect.

220 General discussion and future perspective

Patients without a genetic diagnosis In more than half of the CVI patients in the cohort of the study described in chapter 3 and 4 we did not identify any possible cause. Also in the patients for which we performed WES, some remain without a plausible genetic diagnosis. There are several possible explanations. The first explanation is the coverage of the exonic regions. For example, a frameshift mutation in NR2F1 was missed in one of the patients due to low coverage. Although WES has improved considerably in the last years, the exome coverage of whole genome sequencing (WGS) still outperforms WES.419-421 Moreover, with WGS unbalanced structural variants can be detected more accurately, and (small) balanced structural pathogenic variants can be identified.256 In addition, WGS can also be used to assess non-coding variants, but interpretation of these variants remains difficult. At the moment, however, the costs of the analysis and the large amount of data per genome are the main limitations of the use of WGS in the routine diagnostic setting.419-421 In some of the unsolved patients, the variant might have been picked up by WES analysis, but subsequently not prioritized or wrongly classified. The variant might not have been prioritized when the disorder is caused by digenically inherited or polygenic variants, which can be detected by using a candidate gene based approach.422 If the variant is wrongly classified (e.g. due to the prediction programs, or the GO- and MP-term enrichment), this might be overcome when meta-analysis of large dataset is performed. When variants in a specific gene are enriched in a dataset of patients with CVI compared to controls, this gene is worth to reconsider. Other causes of CVI that might have been missed in the current study are mutations in the mitochondrial DNA or methylation defects. In addition, environmental factors can also be the cause of unsolved cases of CVI; a subclinical prenatal infection, or unrecognized problems during birth. Even more difficult to unravel is a genetic predisposition to be vulnerable for environmental threats, a multifactorial aetiology.

5.4 Final remark

In this thesis we investigated the genetic causes of CVI and have shown that genetic aberrations play an important role in the aetiology. NR2F1 aberrations appeared to be a major cause of CVI, and are associated with Bosch-Boonstra-Schaaf optic atrophy syndrome. Knowledge about the aetiology will improve our understanding of the (mal)function of vision and is nec- Chapter 5 essary to allow subdivision of CVI. This will enable the development of CVI-subtype specific guidance and visual rehabilitation programs. This thesis shows the importance of genetic testing in CVI patients and comprehensive ophthalmological investigation patients with ID.

221

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245 Summary

Summary

Cerebral visual impairment (CVI) is a major cause of low vision in children due to impairment in projection and/or interpretation of the visual input in the brain. The impairment can affect many aspects of vision, and may consist of low visual acuity, visual field defects, motility disorders of the eye and/or higher visual perceptual disorders. The patients are often intellectually disabled, and, vice versa, CVI is present in 5-20% of the patients with an intellectual disability (ID). Other co-morbidities, such as epilepsy, multiple congenital abnormalities or hearing loss, can be present. CVI was first recognized as a consequence of acquired brain damage, mainly due to preterm birth. However, which genetic defects play a role in the aetiology was largely unknown. In chapter 1 an overview of the anatomy and the pre- and postnatal development of the visual system is given, and the features and causes of CVI are described. Higher perceptual deficits with normal visual acuity have been systematically investigated in the past and are present in some genetic disorders. However, CVI with low vision is, in general, only sporadically mentioned in case reports concerning patients with a genetic syndrome. In the last hundred years the knowledge of genetics and the methods to investigate the genome have improved tremendously. Microarray and whole exome sequencing (WES), predominantly to detect copy number variants and single nucleotide variants respectively, are the main techniques to identify genetic aberrations nowadays. To investigate whether and which genetic causes are important in the pathogenesis of CVI, two file studies were performed, presented inchapter 2. Patients with CVI, low vision (visual acuity ≤0.3 and/or visual field <30°), and no major ocular disease (e.g. retinopathy of prematurity) were included. First, 309 CVI patients were analysed for aetiology and ocular variables (chapter 2.1). Almost all, 96% (298/309), had ID. In 60% (184/309) a putative cause was present, such as periventricular leukomalacia or congenital disorder of glycosylation type 1a. In 9% of the individuals (28/309) a genetic disorder was present, and these persons had no other risk factors that could cause CVI (e.g. preterm birth). Moreover, CVI could be associated with ATR-X, Mowat-Wilson, and Pitt Hopkins syndrome for the first time. Comparing the ocular variables in patients with a genetic disorder strabismus (64% versus 88%), pale optic discs (27% versus 65%) and visual field defects (30% versus 72%) could be observed less frequent than in patients with an acquired cause. In chapter 2.2 chromosomal aberrations identified in 607 patients were further classified for pathogenicity. In 7% (41/607) of the patients the chromosomal aberrations were associated with CVI. For four aberrations, present in 26 patients, the association with CVI had been reported before: trisomy 21, 1p36 deletion syndrome, 17p13.3 deletion syndrome (Miller- Dieker syndrome) and 22q13.3 deletion syndrome (Phelan-McDermid syndrome), whereas chromosomal aberrations in another 15 patients had not been reported earlier. In chapter 3 and chapter 4 56 patients without risk factors for CVI were included from 828 persons with CVI and low vision. Of these patients the history was taken, and dysmor-

246 Summary phological, ophthalmological and genetic examinations, mainly microarray and WES, were performed. The results of WES investigations in 25 patients and their parents are reported in chapter 3.1. In addition to CVI, all 25 patients had ID. The WES-data were analysed for de novo, autosomal recessive and X-linked variants, and subsequently, classified into known, candidate or unlikely to be associated with CVI. This classification was based on the Online Mendelian Inheritance in Man database, literature reports, variant characteristics and functional relevance of the gene. After classification, variants in four genes known to be associated with CVI were identified in five patients (20%), establishing a conclusive genetic diagnosis for CVI. In addition, in 11 patients (44%) with CVI, variants in one or more candidate genes were identified. In one patient we could associate compound heterozygous variants in PGAP1 for the first time with CVI, which is further discussed in chapter 3.2. PGAP1 is a member of the glycosylphosphatidylinositol-anchor biosynthesis and remodelling pathway and defects in this pathway form a subclass of congenital disorders of glycosylation. Until that moment two siblings with homozygous PGAP1 variants had been reported, and the only phenotypic feature in common was ID. Therefore, support was obtained for the pathogenicity of variants we identified by the use ofPGAP1 -deficient Chinese hamster ovary cell lines and lymphoblastoid cell lines of the affected individual. Recently, another patient with CVI and PGAP1 deficiency had been reported, which confirms our PGAP1 association with CVI. In chapter 3.3 the identification ofNR2F1 aberrations is described. In the CVI cohort (n=56) three persons had a de novo heterozygous mutation and one individual had a deletion of NR2F1. Moreover, we could recruit two additional patients with de novo NR2F1 aberrations, one of which also had CVI. In addition, all patients had mild to moderate intellectual impairment and optic disc abnormalities, including enlarged excavation and pale and/or small optic discs. NR2F1 encodes a nuclear receptor protein that regulates transcription. A reporter assay showed that NR2F1 transcriptional activity was decreased for the constructs containing the identified missense mutations in the DNA-binding domain and the putative ligand-binding domain. These findings indicated that disruption of NR2F1 could lead to optic atrophy with ID. All identified aberrant genes or chromosomal regions in the 56 persons with CVI are summarized in chapter 4. In 11 (20%) of the 56 persons we could identify a genetic aberration that was previously associated with CVI and in 19 individuals (34%) a potential cause for the CVI was revealed. For the last group we need to identify additional patients with mutations in these genes to establish the causal relationship. In the last chapter, chapter 5, the challenge of the interpretation of genetic variants is discussed. Variant characteristics, functional investigations and recurrence can be helpful in classifying these variants. Because several variants have been identified in genes known to cause ID and many CVI patients have ID, the question was addressed whether CVI is a separate entity. However, it seems that the development of the visual system is not always ppendices A comparable to the cognitive developmental level: it sometimes is more severely affected and

247 Summary sometimes relatively well preserved. To further unravel this and to gain more insight in the pathophysiology, animal models and functional MRI can be used. For these investigations a homogeneous study group is necessary, obtained by a similar underlying acquired or gene defect. Moreover, subdivision of CVI based on aetiology will enable the development of CVI- subtype specific guidance and visual rehabilitation programs.

248 Samenvatting

Samenvatting

Cerebrale visussstoornis (cerebral visual impairment, CVI) is een belangrijke oorzaak van slechtziendheid bij kinderen. CVI is het gevolg van een stoornis in projectie en/of interpretatie van de visuele input in de hersenen. Verschillende aspecten van het zien kunnen hierdoor aangedaan zijn en dit kan leiden tot bijvoorbeeld een lage gezichtsscherpte, beperkt gezichtsveld, oogbewegingsstoornissen of problemen met de visuele perceptie. De patiënten zijn vaak verstandelijk beperkt en omgekeerd heeft 5-20% van de patiënten met een verstandelijk beperking (VB) een CVI. Andere co-morbiditeiten, zoals epilepsie, multipele aangeboren afwijkingen of gehoorsverlies kunnen aanwezig zijn. CVI werd in eerste instantie herkend bij personen met verworven hersenschade, vaak ten gevolge van vroeggeboorte. Echter welke genetische afwijkingen een rol spelen in de etiologie was grotendeels onbekend. In hoofdstuk 1 wordt een overzicht gegeven van de anatomie en de pre- en postnatale ontwikkeling van het visuele systeem. Daarnaast worden de kenmerken en oorzaken van CVI beschreven. In de literatuur is van sommige genetische aandoeningen systematisch onderzocht of hogere perceptuele stoornissen met een normale gezichtsscherpte aanwezig zijn. Echter, CVI met een lage gezichtscherpte ten gevolge van een genetische aandoening is over het algemeen slechts sporadisch genoemd in casuïstiek. De kennis van de genetica en de technieken om genetisch onderzoek te verrichten zijn in de laatste eeuw sterk ontwikkeld. “Microarray” en “whole exome sequencing” (WES), met name om chromosoomafwijkingen en nucleotide varianten te detecteren, zijn tegenwoordig de voornaamste technieken om genetische afwijkingen vast te stellen. In dit onderzoek zijn met behulp van deze technieken de genetische oorzaken in CVI-cohort met lage gezichtsscherpte in kaart gebracht. In hoofdstuk 2 worden de twee dossier-onderzoeken beschreven die zijn verricht. Daarin is onderzocht of en welke genetische oorzaken belangrijk zijn in de pathogenese van CVI. Patiënten met CVI, slechtziendheid (gezichtsscherpte ≤0.3 of gezichtsveld <30°) en geen belangrijke oogziekte (bijv. prematuren retinopathie) werden geïncludeerd. In het eerste dossier-onderzoek werden bij 309 CVI-patiënten de etiologie en de bevindingen bij oogheelkundig onderzoek geanalyseerd (hoofdstuk 2.1). Bijna alle patiënten, 96% (298/309), had een VB. In 60% van de patiënten (184/309) was een mogelijke oorzaak te identificeren, zoals periventriculaire leukomalacie of een congenitale glycosyleringsstoornis type 1a. In 9% van de personen (28/309) was een genetische aandoening vastgesteld zonder dat andere risicofactoren die CVI kunnen veroorzaken (zoals vroeggeboorte) aanwezig waren. Daarnaast konden we CVI voor de eerste keer associëren met ATR-X, Mowat-Wilson, en Pitt Hopkins-syndroom. Vervolgens werden de bevindingen bij oogheelkundig onderzoek vergeleken tussen de patiënten met een genetische aandoening en die met een verworven ppendices A oorzaak. Strabismus (64% versus 88%), een bleke papil (27% versus 65%) en een beperkt

249 Samenvatting gezichtsveld (30% versus 72%) kwamen vaker voor bij de patiënten met een verworven oorzaak. In het tweede dossier-onderzoek (hoofdstuk 2.2) werden de gevonden chromosoomafwijkingen in 607 patiënten verder geclassificeerd op pathogeniciteit. Bij 7% (41/607) van de patiënten werd een chromosoomafwijking geassocieerd met CVI. Bij vier chromosoomafwijkingen, aanwezig in 26 patiënten, was de associatie met CVI eerder gepubliceerd: trisomie 21, 1p36 deletie-syndroom, 17p13.3 deletie-syndroom (Miller-Dieker-syndroom) en 22q13.3 deletie-syndroom (Phelan-McDermid-syndroom), terwijl de chromosoomafwijkingen in 15 andere patiënten nog niet eerder beschreven waren in relatie tot CVI. Voor hoofdstuk 3 en hoofdstuk 4 werden uit 828 personen met CVI en slechtziendheid 56 patiënten zonder risicofactoren voor CVI geïncludeerd. Een uitgebreide anamnese werd afgenomen en dysmorfologisch, oogheelkundig en genetisch onderzoek werd verricht. Het genetische onderzoek bestond voornamelijk uit microarray en WES. De resultaten van het WES-onderzoek in 25 patiënten en hun ouders is beschreven in hoofdstuk 3.1. Naast CVI hadden alle 25 patiënten een VB. De WES-data is geanalyseerd voor varianten die een de novo, autosomaal recessief, en X-gebonden recessieve overerving volgen. Vervolgens zijn de varianten geclassificeerd in varianten met een bekende, kandidaat- of onwaarschijnlijke associatie met CVI. Deze classificatie is gebaseerd op de Online Mendelian Inheritance in Man database, literatuur, variant-karakteristieken en functionele relevantie van het gen. In vier verschillende genen die een bekende associatie met CVI hadden, werden in vijf personen (20%) varianten gevonden. Dit leidde tot een sluitende genetische diagnose voor CVI. Daar- naast werden in 11 patiënten (44%) varianten in een of meerdere kandidaat-genen geïdenti- ficeerd. In één patiënt konden we een nieuwe associatie leggen tussen CVI en de gevonden compound heterozygote varianten in PGAP1. Dit wordt verder besproken in hoofdstuk 3.2. PGAP1 is een onderdeel van de glycosylphosphatidylinositol-anker-biosynthese en de remodelleringsroute. Defecten in deze biosynthese of route vormen een subklasse van congenitale glycosyleringsstoornissen. Destijds waren er twee families met kinderen met homozygote PGAP1-varianten gerapporteerd en het enige gezamenlijke kenmerk was VB. Daarom werd extra bewijs verkregen voor de pathogeniciteit van de door ons gevonden varianten door middel van eicellijnen van de Chinese hamster en lymfoblastoïde cellijnen van de patiënt. Recentelijk werd een andere patiënt met CVI en PGAP1-deficiëntie beschreven hetgeen onze associatie met CVI bevestigt. In hoofdstuk 3.3 wordt de ontdekking van NR2F1-afwijkingen beschreven. In het CVI- cohort (n=56) hadden drie personen een de novo heterozygote mutatie en één patiënt een deletie van NR2F1. Er werden nog twee patiënten met de novo NR2F1-afwijkingen geïdentifi- ceerd (buiten het CVI-cohort), van wie één ook CVI bleek te hebben. Alle 6 patiënten hadden milde tot matige VB en papil-afwijkingen, waaronder een vergrote excavatie en een bleke of kleine papil. NR2F1 codeert voor een nucleair receptor-eiwit dat de transcriptie reguleert. Een reporter-assay toonde aan dat de transcriptionele activiteit van NR2F1 was verminderd voor

250 Samenvatting de constructen die de gevonden missense mutatie bevatten, welke gelokaliseerd waren in het DNA-bindings domein en in het mogelijke ligand-bindings domein. Deze bevindingen wijzen erop dat een verstoring van NR2F1 kan leiden tot opticus atrofie met VB. Alle gevonden afwijkende genen en chromosomale regio’s in de 56 personen met CVI zijn samengevat in hoofdstuk 4. In 11 (20%) van de 56 personen konden we een genetische afwijking vinden die eerder geassocieerd was met CVI en in 19 patiënten (34%) konden we een mogelijke oorzaak voor de CVI detecteren. Voor deze laatste groep zijn meer patiënten met mutaties in hetzelfde gen nodig om een oorzakelijk relatie vast te stellen. In het laatste hoofdstuk, hoofdstuk 5, worden de genetische variant interpretatie en de bijbehorende uitdagingen bediscussieerd. Variant-karakteristieken, functionele onderzoeken en meerdere personen met een variant in het hetzelfde gen kunnen behulpzaam zijn in het beoordelen van de varianten. Omdat verschillende varianten gevonden werden in genen die geassocieerd zijn met VB en vele CVI patiënten een VB hebben, wordt de vraag besproken of CVI een aparte aandoening is. Echter, het lijkt dat de ontwikkeling van het visuele functioneren niet altijd vergelijkbaar is met het cognitieve niveau; soms loopt de ontwikkeling van het visuele functioneren voor en soms achter ten opzichte van de cognitieve ontwikkeling. Om meer inzicht te krijgen in de onderliggende pathofysiologie kunnen diermodellen en functionele MRI worden gebruikt. Voor deze onderzoeken zijn homogene studiegroepen nodig, welke kunnen worden verkregen door patiënten te verzamelen met eenzelfde verworven of genetische oorzaak. Bovendien opent het onderverdelen van CVI gebaseerd op etiologie de mogelijkheid om begeleiding en therapie te ontwikkelen die specifiek is voor het subtype CVI. ppendices A

251 List of authors and affiliations

List of authors and affiliations

Ellen A.W. Blokland Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands

F. Nienke Boonstra Bartiméus, Institute for the Visually Impaired, Zeist, The Netherlands Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Radboud university medical center, Nijmegen, the Netherlands

Daniëlle G.M. Bosch Bartiméus, Institute for the Visually Impaired, Zeist, The Netherlands Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Radboud university medical center, Nijmegen, the Netherlands

Chun-An Chen Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, USA

Frans P.M. Cremers Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands

Richard A. Gibbs Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Human Genome Sequencing Center, Baylor College of Medicine, Houston, USA

Christian Gilissen Department of Human Genetics, Radboud university medical center, Nijmegen, the Nether- lands

252 List of authors and affiliations

Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, the Netherlands

Claudia Gonzaga-Jauregui Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA

Shalini Jhangiani Human Genome Sequencing Center, Baylor College of Medicine, Houston, USA

Taroh Kinoshita Research Institute for Microbial Diseases and WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan

Nicole de Leeuw Department of Human Genetics, Radboud university medical center, Nijmegen, the Nether- lands

Richard A. Lewis Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Texas Children’s Hospital, Houston, USA Department of Ophthalmology, Baylor College of Medicine, Houston, USA Departments of Pediatrics, Baylor College of Medicine, Houston, USA

Joep de Ligt Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands Institute for Genetic and Metabolic Disease, Radboud university medical center, Nijmegen, The Netherlands Hubrecht Institute-KNAW, University Medical Centre Utrecht, CancerGenomics.nl, Utrecht, the Netherlands

James R. Lupski Human Genome Sequencing Center, Baylor College of Medicine, Houston, USA Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Texas Children’s Hospital, Houston, USA

Departments of Pediatrics, Baylor College of Medicine, Houston, USA ppendices A

253 List of authors and affiliations

Yoshiko Murakami Research Institute for Microbial Diseases and WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan

Donna M. Muzny Human Genome Sequencing Center, Baylor College of Medicine, Houston, USA

Willy M. Nillesen Department of Human Genetics, Radboud university medical center, Nijmegen, the Nether- lands

Rolph Pfundt Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands

Margot R.F. Reijnders Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Radboud Institute for Health Sciences, Radboud university medical center, Nijmegen, The Netherlands

Christian P. Schaaf Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Texas Children’s Hospital, Houston, USA Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, USA

Liesbeth Spruijt Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Research Institute for Oncology, Radboud university medical center, Nijmegen, The Nether- lands

Ming-Jer Tsai Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, USA

Sophia Y. Tsai Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, USA

254 List of authors and affiliations

Lisenka E.L.M. Vissers Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands Institute for Genetic and Metabolic Disease, Radboud university medical center, Nijmegen, The Netherlands

Bert B.A. de Vries Department of Human Genetics, Radboud university medical center, Nijmegen, The Nether- lands Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Radboud university medical center, Nijmegen, the Netherlands Radboud Institute for Health Sciences, Radboud university medical center, Nijmegen, The Netherlands

Michèl A.A.P. Willemsen Donders Institute for Brain, Cognition and Behavior, Radboud university medical center, Nijmegen, The Netherlands Department of Pediatric Neurology, Radboud university medical center, Nijmegen, The Netherlands

Wojciech Wiszniewski Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Texas Children’s Hospital, Houston, USA

Mafei Xu Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, USA

Helger G. Yntema Department of Human Genetics, Radboud university medical center, Nijmegen, The Netherlands

Huda Y. Zoghbi Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA Departments of Pediatrics, Baylor College of Medicine, Houston, USA Howard Hughes Medical Institute, USA Departments of Neuroscience, Program in Developmental Biology, Baylor College of Medi- cine, Houston, USA ppendices A Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, USA

255 Dankwoord/Acknowledgements

Dankwoord/Acknowledgements

Het is af, vier jaar werken aan één doel. Nu nog een dankwoord, en zowaar bijna helemaal in het Nederlands. Kort samengevat: het was een top tijd, mede mogelijk gemaakt door velen. Iedereen die heeft bijgedragen aan dit proefschrift, direct of indirect, wil ik van harte bedanken. Verschillende mensen wil ik echter apart bedanken. Te beginnen met de patiënten en hun ouders. Zonder hen was dit onderzoek niet mogelijk geweest.

Natuurlijk mijn co-promotoren Nienke Boonstra en Bert de Vries, en promotor Frans Cremers; bedankt voor jullie ondersteuning, zinvolle discussies en kritische vragen. Nienke, dankzij jou ben ik wegwijs geworden in de oogheelkundige wereld en leerde ik dat er een hele andere denkwijze is tussen genetici en oogartsen. Met bewondering zag ik hoe je rustgevend zingend het netvlies van een ernstige verstandelijke patiënt inspecteerde. Bert, jouw tomeloze enthousiasme zorgde ervoor dat ik niet vergat te genieten van de mooie resultaten. Hoewel het soms even slikken was als een artikel, poster of presentatie weer eens rood doorstreept terugkwam, werd het uiteindelijk wel een stuk beter. Frans, mijn onderzoeksfocus lag achter de retina, ook voor jou minder bekend terrein. Toch gaf je mij de mogelijkheid me helemaal te verdiepen in CVI. Jouw moleculaire kennis en gestructureerde en precieze werkwijze was van grote waarde.

Veel betrokken was er natuurlijk van de zijde van het Bartiméus. Piet ondanks je pensioen heb je vol enthousiasme meegeholpen tijdens de oogheelkundige onderzoeken. Ook de andere orthoptisten, Ellen, Lia, Marjoke, Amir, Alexander, Inge, Florine, Thekla, Heleen en Nardi, de klinisch fysici, Frans, Frank en Gerard en oogartsen, José, Mies en Ymkje, hartelijk dank voor jullie ondersteuning. Dank natuurlijk ook aan Helga, Karin, Ineke en Antoinette voor de eerste opvang van bel- lende ouders en allerlei administratieve zaken.

Aan mijn mede-onderzoekers in Bartiméus. Mieke, Joyce, Bianca, Annemieke en Annemieke. Fijn om het ‘lief en leed’ te kunnen delen in de soms te kleine en nu opgeheven ‘onderzoeks- kamer’.

Natuurlijk is er ook in het Radboudumc een hoop ‘lief en leed’ gedeeld met mijn kamergenoten. Anneke wat heerlijk om met een “senior” promovendus op de kamer te zitten aan wie je alles kunt vragen. Margot, ineens was ik de “senior” promovendus, maar niks is zo leuk als meedenken over andermans onderzoek. Leonie, ook al was het maar kort, wat een gezellig- heid! Willemijn, jij had altijd tips waar de interessante praatjes zijn.

256 Dankwoord/Acknowledgements

Mijn huidige kamergenoten, Trudy, Rita, Ronald en Kees, en alle andere collega’s bij de ge- noomdiagnostiek, een arts in het lab, jullie durfden het aan. Van mijn kant bevalt het goed, ook al het gebak!

Helger, Rolph, Nicole, Lisenka, Arjan, Willy en Tuula, tijdens mijn onderzoek dachten jullie actief mee, onder andere tijdens het ID overleg, en leerde ik een hoop over array-afwijkingen en WES-interpretatie.

Ellen en Marijke, jullie maakten me wegwijs in het research lab. Van jullie leerde ik een gel gieten en het doen van een PCR. Irene, een lab verder, bedankt voor het leren van de MAQ assay. Anna, we analysed together my first exome results. It seemed to be such a clear case... To the others of the blindness genetics group, thanks for your help and good cheer.

Christian, Joep, Nienke, Rick en alle andere bioinformatici, dankzij jullie prachtige pijplijn kon ik de data analyseren.

Aisha, Arjen, Gijsbert en Ilja, Janneke, Lot, Tjitske, Thatjana en Serwet , Corrie, het secretariaat en klinische genetici: fijn dat ik jullie af en toe kon storen voor inhoudelijke en logistieke vragen, maar ook bedankt voor de ontspannende lunches op de patio.

Vrienden en (schoon-)familie: jullie zorgden voor afleiding van het werk, leuke dingen doen en ontspannen!

Sandra en Martijn, jullie staan vandaag aan mijn zijde als paranimf. Sandra, petje af hoe jij alles combineert. Heel veel succes met het jouw onderzoek. Over een paar jaar sta jij in het midden. Martijn, ook al ligt jouw expertise heel ergens anders, je bent altijd in voor een kritische en nuttige discussie over genetica. Fijn dat je naar Nederland bent gekomen voor deze dag. Je bent een broer waar je op kunt bouwen!

Pap en mam, jullie hebben me altijd gesteund en de mogelijkheid geboden om mijn eigen weg te zoeken. Ik kan jullie altijd bellen voor tips en adviezen. Ontzettend fijn dat jullie om de week komen oppassen en en passant nog wat klusjes in huis doen.

Lieve Floor, een knuffel of een lach maakt alles goed en relativeert een hoop. Nu is er tijd voor gezinsuitjes naar het zwembad of de speeltuin in plaats van naar de drukker (al was de licht- gevende drone van de drukker ook wel erg interessant). Peter, we hebben het gehaald! Jij weet als geen ander wat het is om te promoveren, al was ons promotietraject en onderzoek totaal verschillend. Nu jij alles weet van uitbesteding, moeten we dat misschien ook maar ppendices A eens gaan doen.423

257 Curriculum Vitae

Curriculum Vitae

Daniëlle Gerda Maria Bosch was born on the 10th of June 1983 in Tegelen. In 2001 she gradu- ated from grammar school ‘Den Hulster’ in Venlo, and started as a medical student at the Radboud University in Nijmegen. During her student days, she founded the student jujutsu association ‘Zanshin’, co-organized a science day for the medical faculty, and co-organized the Batavierenrace twice. The Batavierenrace is the largest student relay race in the world. During the 33rd Batavierenrace, Daniëlle had a part-time function. For the 34th Batavieren- race, however, she interrupted her studies to become a full-time board member (secretary) of the organizing committee. Afterwards, she began with her internships in July 2006. Her final internship was in Tanzania, where she worked in a rural hospital for three months. After practising basic medicine, Daniëlle did her research rotation concerning aneuploidy of chromosome 21 at the department of Human Genetics in Nijmegen, with data collected using a state of the art technique, microarray. In 2008 she obtained her medical degree and worked at the Neurology department in ‘Ziekenhuis Gelderse Vallei’ from February 2009 until February 2010. Subsequently, Daniëlle worked for 18 months at the Clinical Genetics department at Erasmus MC, before starting her PhD project in September 2011. The project concerned the clinical and genetic aspects of cerebral visual impairment and was executed at Bartiméus and the Human Genetics department of the Radboud university medical center (Thesis supervisor: prof. dr. F.P.M. Cremers. Co-supervisors: mrs. dr. F.N. Boonstra and dr. B.B.A. de Vries). She presented her research at several national and international meetings, and was awarded with the poster award at the European Human Genetics Conference 2014 for her poster ‘NR2F1 mutations cause optic atrophy with intellectual disability’. In August 2015 she started working at the Genome Diagnostics division of the department of Human Genetics at the Radboud university medical center in Nijmegen. In 2016 she will start working at the Genetics department in UMC Utrecht. In her spare time, Daniëlle plays the saxophone in ‘Harmonie St. Cecilia’ and Noviomagum Wind Orchestra (NMWO). She was a board member of NMWO between 2012 and 2014. She is married to Peter Laaper and has one daughter, Floor.

258 List of publications

List of publications

De novo mutations of RERE cause a new genetic syndrome whose features overlap those associated with 1p36 deletions Fregeau, B., Kim, B.J., ,Hernandez, A., Jordan, V.K., Cho, M., GeneDx authors,4 , Rosenfeld, J.A., Bhoj, E., Sacharow, S., Baranano, K., Bosch, D.G., de Vries, B.B., van Haelst, M., Lindstrom, K., James, P., Kulch, P. Scott, D.A.,* Sherr, E.H.* Submitted.

The expanding clinical phenotype of Bosch-Boonstra-Schaaf Optic Atrophy syndrome: 20 new cases, and possible genotype-phenotype correlations Chen, C.A.,* Bosch, D.G.,* Cho, M.T., Rosenfeld, J.A., Shinawi, M., Lewis, R.A., Mann, J., Jayakar, P., Payne, K., Walsh, L., Moss, T., Schreiber, A., Schoonveld, C., Monaghan, K.G., Elmslie, F., Doug- las, G., Boonstra, F.N., Millan, F., Cremers, F.P., McKnight, D., Richard, G., Juusola, J., Kendall, F., Ramsey, K., Anyane-Yeboa, K., Malkin, E., Chung, W.K., Niyazov, D., Pascual, J.M., Walkiewicz, M., Veluchamy, V., Li, C., Hisama F.M., de Vries, B.B.,† Schaaf, C.P.† Submitted.

TRIO loss of function is associated with mild intellectual disability and affects dendritic branching and synapse function Ba, W.,* Yan, Y.,* Reijnders, M.R.,* Feenstra, I., Bongers, E.M., Bosch, D.G., de Vries, P.F., Veltman, J.A., Hoischen, A., Mefford, H.C., Eichler, E.E., Eipper, B., Mains, R., Vissers, L.E., Nadif Kasri, N.,† de Vries, B.B.† Submitted.

Putative digenic inheritance of heterozygous RP1L1 and C2orf71 null mutations in syndromic retinal dystrophy Liu, Y.P.,* Bosch, D.G.,* Siemiatkowska, A.M., Rendtorff, N.D., Boonstra, F.N., Möller, C.M. Tranebjærg, L., Katsanis,N.,† Cremers, F.M.† Submitted.

De novo loss-of-function mutations in WAC cause a recognizable intellectual disability syndrome and are associated with learning deficits in Drosophila Lugtenberg, D.,*, Reijnders, M.R.,* Fenckova, M.,* Bijlsma, E.K., Bernier, R., van Bon, B.W., Smeets, E., Vulto-van Silfhout, A.T., Bosch, D.G., Eichler, E.E., Mefford, H.C., Carvill, G.L., Bongers, E.M., Schuurs-Hoeijmakers, J.H., Ruivenkamp, C.A., Santen,G.W., van den Maagdenberg, A.M., Peeters-Scholte, C.M., Kuenen, S., Verstreken, P., Pfundt, R., Yntema, H.G., de Vries, P.F., Velt- † † † man, J.A., Hoischen, A., Gilissen, C., de Vries, B.B., Schenck, A., Kleefstra, T., Vissers, L.E. ppendices A Eur J Hum Genet. Accepted.

259 List of publications

Novel genetic causes for cerebral visual impairment Bosch, D.G., Boonstra, F.N., de Leeuw, N., Pfundt, R., Nillesen, W.M., Ligt, J., Gilissen, C., Jhangi- ani, S., Lupski, J.R., Cremers, P.M., de Vries, B.B. Eur J Hum Genet. Epub ahead of print. http://www.ncbi.nlm.nih.gov/pubmed/26350515

Cerebral visual impairment and intellectual disability caused by PGAP1 variants. Bosch, D.G., Boonstra, F.N., Kinoshita, T., Jhangiani, S., de Ligt, J., Cremers, F.P., Lupski, J.R., Murakami, Y., and de Vries, B.B. Eur J Hum Genet 2015; 23: 1689-1693. http://www.ncbi.nlm.nih.gov/pubmed/25804403

Cerebral visual impairment, autism, and pancreatitis associated with a 9 Mbp deletion on 10p12. Bosch, D.G., Boonstra, F.N., Pfundt, R., Cremers, F.P., and de Vries, B.B. Clin Dysmorphol 2015; 24: 34-37. http://www.ncbi.nlm.nih.gov/pubmed/25356883

Age-related decreased inhibitory vs. excitatory gene expression in the adult autistic brain. van de Lagemaat, L.N., Nijhof, B.,* Bosch, D.G.,* Kohansal-Nodehi, M.,* Keerthikumar, S.,* and Heimel, J.A. Front Neurosci 2014; 8: 394. http://www.ncbi.nlm.nih.gov/pubmed/25538548

Chromosomal aberrations in cerebral visual impairment. Bosch, D.G., Boonstra, F.N., Reijnders, M.R., Pfundt, R., Cremers, F.P., and de Vries, B.B. Eur J Paediatr Neurol 2014; 18: 677-684. http://www.ncbi.nlm.nih.gov/pubmed/24912731

Low vision due to cerebral visual impairment: differentiating between acquired and genetic causes. Bosch, D.G., Boonstra, F.N., Willemsen, M.A., Cremers, F.P., and de Vries, B.B. BMC Ophthalmol 2014; 14: 59. http://www.ncbi.nlm.nih.gov/pubmed/24886270

Nonpenetrance of the most frequent autosomal recessive leber congenital amaurosis mutation in NMNAT1. Siemiatkowska, A.M., Schuurs-Hoeijmakers, J.H., Bosch, D.G., Boonstra, F.N., Riemslag, F.C., Ruiter, M., de Vries, B.B., den Hollander, A.I., Collin, R.W., and Cremers, F.P. JAMA ophthalmology 2014; 132: 1002-1004. http://www.ncbi.nlm.nih.gov/pubmed/24830548

NR2F1 mutations cause optic atrophy with intellectual disability. Bosch, D.G.,* Boonstra, F.N.,* Gonzaga-Jauregui,* C., Xu, M., de Ligt, J., Jhangiani, S., Wiszniewski, W., Muzny, D.M., Yntema, H.G., Pfundt, R., Vissers, L.E., Spruijt, L., Blokland, E.A.,

260 List of publications

Chen, C.A., Baylor-Hopkins Center for Mendelian Genomics, Lewis, R.A., Tsai, S.Y., Gibbs, R.A., Tsai, M.J., Lupski, J.R., Zoghbi, H.Y., Cremers, F.P.,† de Vries, B.B.,† and Schaaf, C.P.† Am J Hum Genet 2014; 94: 303-309. http://www.ncbi.nlm.nih.gov/pubmed/24462372

Brachydactyly: a rare complication of sickle cell anaemia. Bosch, D.G., van Nieuwenhoven, C.A., and Hoogeboom, A.J. Clin Dysmorphol 2011; 20: 172-173. http://www.ncbi.nlm.nih.gov/pubmed/21546827

*These authors contributed equally to this work. †These authors contributed equally to this work and are co-senior authors. ppendices A

261 List of abbreviations

List of abbreviations

ALP alkaline phosphatase array CGH array-based comparative genomic hybridization ATR-X alpha-thalassemia X-linked intellectual disability CADD Combined Annotation Dependent Depletion CDG congenital disorder of glycosylation CHO Chinese hamster ovary CMV cytomegalovirus CNV copy number variants CVI cerebral visual impairment CSWS continuous spike and wave during slow wave sleep DBD DNA-binding domain DGV Database of Genomic Variants. DVM delayed visual maturation EEG electroencephalography ERG electroretinography FISH fluorescence in situ hybridization fMRI functional MRI GO-term gene ontology term GPI glycosylphosphatidylinositol GPI-AP GPI-anchored proteins ID intellectual disability IRB institutional review board HGMD human gene mutation database LBD ligand-binding domain LCA Leber congenital amaurosis LCL lymphoblastoid cell lines LGN lateral geniculate nucleus LH charts Lea Hyvarinen charts LOVD Leiden open variation database MIP multiplex inversion probe MLPA multiplex ligation-dependent probe amplification MP-term mouse phenotype term MRI magnetic resonance imaging NGS next generation sequencing OCT optical coherence tomography OMIM Online Mendelian Inheritance in Man PI-PLC phosphatidylinositol-specific phospholipase C

262 List of abbreviations

PVL periventricular leukomalacia ROP retinopathy of prematurity TAC Teller acuity cards VA visual acuity VEP visual evoked potential WES whole exome sequencing WGS whole genome sequencing WT wild-type ppendices A

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Cerebral visual impairment from clinic to genetics Daniëlle G.M. Bosch