ELECTROPHYSIOLOGY IN VISUALLY IMPAIRED CHILDREN … · Introduction 1.1 Visual impairment in Dutch children The prevalence of childhood visual impairment in Northern en Western Europe,

Electrophysiology in visually

impaired children

ELECTROPHYSIOLOGY IN VISUALLY IMPAIRED CHILDREN MARIA MICHIELDE VAN GENDEREN

Copyright © Maria Michielde van Genderen

Uitgave: Universiteit Utrecht

Druk: Febo Druk b.v.

Boekverzorging: Quasi Grafische producties (Mirjam Roest)

NUR: 681

ISBN-10: 90-393-4244-X

ISBN-13: 978-90-393-4244-2


Electrophysiology in visually

impaired children

Elektrofysiologisch onderzoek bij kinderen

met een visuele beperking

(met een samenvatting in het Nederlands)

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. W.H. Gispen, ingevolge

het besluit van het college voor promoties in het openbaar teverdedigen op dinsdag 23 mei 2006 des middags te 4.15 uur

door

Maria Michielde van Genderen

geboren op 29 juni 1959 te Utrecht


Promotoren: Prof. dr. J.S. StilmaProf. dr. F.M. Meire

Co-promotor: dr. F.C.C. Riemslag

Dit proefschrift werd mogelijk gemaakt met financiële steun van de Vereniging Bartiméus


Voor mijn ouders,voor Michiel en Menno



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CONTENTS

Chapter 1

General introduction 9

Chapter 2

The key role of electrophysiology in the diagnosis of visually impaired children 25

Chapter 3

Thiamine-responsive megaloblastic anemia syndrome (TRMA) with cone-rod dystrophy 45

Chapter 4

Ocular features in Rubinstein-Taybi syndrome: investigation of 24 patients and review of the literature 59

Chapter 5

The EEM syndrome, a ten year follow-up of two new patients 81

Chapter 6

Misrouting and foveal hypoplasia without albinism 95

Chapter 7

Mutations in GRM6 cause autosomal recessive congenital stationary nightblindness with a distinctive scotopic 15-Hz flicker electroretinogram 107

Chapter 8

Summary and recommendations 129

Chapter 9

Samenvatting (Dutch summary) 137

Dankwoord 146Curriculum vitae 148



Chapter 1

General introduction



Introduction

1.1 Visual impairment in Dutch children

The prevalence of childhood visual impairment in Northern en Western Europe,based on the WHO (World Health Organization) definitions1 (see Table 1) isbetween 0.48 and 1.6 per 1000 children.2-4 Childhood blindness has a prevalenceof 0.1 to 0.59 per 1000 children.5,6 The main causes of visual loss in children in thehighly industrialised countries are lesions of the higher visual pathways, opticatrophy and retinal disorders. The most frequent retinal abnormalities arehereditary retinal dystrophies and retinopathy of prematurity.2,3

The concept of what constitutes visual impairment in childhood has changed overthe last 30 years.4,6 There has been increasing recognition of visual impairment inchildren with coexisting neurological disorders, leading to a relative decrease inthe number of children with an isolated visual problem. This change is alsoreflected in the reported prevalence of the various causes of visual impairment. Inolder studies, congenital cataract and retinal disorders were considered to be themost important causes of visual handicap,7,8 while in more recent studies cerebralvisual impairment and optic atrophy are the leading causes.2,9

Exact data on the prevalence of childhood visual impairment in The Netherlandsare not known. Retinal dystrophies, congenital cataracts, and optic nerve abnor-malities were the leading causes of visual impairment in a study by Loewer-Siegerin 19758 and Van der Pol in 1986.10 However, both studies were conducted amongpupils of the Dutch institutions and special schools for visually handicappedchildren. Children with severe mental disability, who have an increased risk of

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level of visual impairment category of vision visual acuity in better eye with optical correction

visual impairment low vision worse than 6/18 (0.3) up to 6/60 (0.1)severe visual impairment low vision worse than 6/60 (0.1) up to 3/60 (0.05)blind blindness worse than 3/60 (0.05) up to no light

perception or visual field ≤ 10ºaround central fixation

Table 1. WHO classification of levels of visual impairment


cerebral visual impairment and optic atrophy11, 12 constituted only a small part ofthe study group. Bartiméus is one of the Dutch institutes that provide rehabilitation, care, andeducation for visually impaired children. Nowadays, approximately half of the re-ferred children are mentally as well as visually impaired, reflecting the prevalenceof multiple handicaps among visually impaired children in general.9 Consequently,at Bartiméus we may expect similar causes of visual impairment as described inrecent studies in other Northern and Western European countries.

1.2 Visual electrophysiology

Visual electrophysiology includes the electro-oculogram (EOG), the electroret-inogram (ERG), and the visual evoked potential (VEP), which assess the functionof the retinal pigment epithelium, retina, and the postretinal pathway to the visualcortex, respectively. The EOG is only of minor importance in the diagnosis ofvisually impaired children,13 and therefore will not be further discussed.

1.2.1. ERG

The Ganzfeld ERG is recorded after pupil dilatation and corneal anaesthesia;electrodes at the cornea record the electrical response of the retina to light. The ISCEV (International Society of Clinical Electrophysiology of Vision)recommends to record at least five standard responses:14, 15

In a dark adapted eye:1. ERG to a dim white flash, arising from the rods;2. ERG to standard white flash (mixed cone-rod response)3. Oscillatory potentials to standard flashes;In a light adapted eye:4. ERG to a standard flash, arising from the cones;5. ERGs to 30 Hz flicker of a standard flash (cone-mediated).

The recommended duration of dark adaptation is at least 20 minutes, of lightadaptation at least 10 minutes. For light adaptation, we use a white 30 cd/m2 rodsaturating background. The standard flash is defined as 1.5-3.0 photopic cd•s•m-2 at the surface of a Ganzfeld bowl with a maximum duration of 5 milli-seconds. The dim white flash has an intensity of -2.6 log of the standard flash. The ERG waveform is an integrated mass response made up of a number ofindependent components.

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The a-wave (see Figure 1) is the first major negative component and is caused byhyperpolarization of the photoreceptors. The positive b-wave results from activityof bipolar cells, the inner plexiform layer and the Müller cells. The amplitude andlatency of the a- and b-wave change with stimulus intensity. Oscillatory potentials(OPs) are rhythmic wavelets on the ascending limb of the b-wave; they probablyare generated between the inner and outer plexiform layers.16 They are measuredwith the use of a bandpass filter between 100 and 200 Hz.

In our department, ERGs are performed without sedation or anaesthesia. We useDTL (Dawson, Trick, and Litzkow)17 fibre electrodes in almost all children. Onlyin a few cases contact lens electrodes are used, mainly in infants. In addition to thestandard ISCEV series, we use an extended series of stimuli in the dark adaptedcondition: -2.0, -1.6, -1.0, -0.6, -0.3, and +0.6 log to the standard. In the lightadapted condition we add +0.6 log to the standard ISCEV intensity. Theadvantage of the extended series is the improved evaluation of the consistency of the standard ISCEV responses; an example in a normal subject is shown inFigure 2.

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Figure 1. Mixed ERG of a normal subject.


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Figure 2. ERG responses of a normal subject, with an extended stimulus series (grey lines) to the standard ISCEV(black lines). The third standard ISCEV response (50 µV, 0.0 ND) consists of OPs.

In cases of suspected congenital stationary night blindness (CSNB), we performmeasurements according to two special protocols, in addition to the extendedstandard series: a "Miyake protocol" , which consists of 30 Hz photopic measure-ments every minute for ten minutes after turning on the background illumination,and 15 Hz scotopic measurements. With the Miyake protocol, the complete andincomplete forms of CSNB (CSNB1 and 2) can be distinguished.18 With the 15 Hzscotopic measurements additional differentiation may be made between X-linkedand autosomal recessively inherited CSNB; this will be further discussed inChapter 7. Technical measures to optimize the reliability of the transiently recordedresponses are on-line rejection of unreliable single ERG responses and fixationmonitoring; these measures will be discussed in detail in Chapter 2. We determined normal values for ERG amplitudes and latencies in 51 children(mean age 10 ± 4 years). Scotopic and photopic b-wave amplitudes are shown inChapter 2, Table 1.

Several studies describe the maturation of ERG and VEP responses with age.19-23

Fulton et al investigated the developmental changes of the ISCEV rod, maximal,and cone responses in normal infants and children.20, 23 They cautioned againstdrawing definite conclusions from absent or very low ERG responses in infants,because until 5 weeks of age, a quarter of the normal infants did not have adetectable rod response. The study also provided expected limits of normal valuesin the first year of life. In evaluating the responses of infants and young children,we take these maturation effects into account.

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1.2.2. VEP

In our department, we perform standard VEPs with seven active occipitalelectrodes (see Figure 3). Two differential recordings O1-O2 and T5-T6 enhanceand visualize the hemispheric differences. Additionally, a Laplacian derivation overthe striate cortex amplifies the contribution of the striate source relative to theextrastriate source.24 In this derivation the responses from four surroundingelectrodes are subtracted from the central one. Because contributions from distantsources in the five electrode positions are correlated, they will be cancelled by thecalculation. Those that are locally important at the central electrode will beenhanced selectively. For VEP stimulation, we use transient flashes, and computer generated checker-board patterns that are presented on a television screen. In pattern reversal VEP,these checkerboards reverse from black to white; in pattern onset VEP they appear

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Figure 3. Schematic representation of the position of the electrodes used for VEP. Seven active occipital electro-des are referenced to a frontal midline electrode. The active midline electrode is placed 2 cm above the inion, withtwo lateral active electrodes on each side, at a spacing of 3 cm. These electrode positions are equivalent to T5, O1,Oz, O2, and T6 of the international 10-20 system. The differential recordings are O1 – O2 and T5 – T6.The Laplacian is given by:Laplacian= Oz – 1/4*(O1+↓inion+O2+Pz)=1/4* ((Oz-O1)+(Oz-↓inion)+(Oz-O2)+(Oz-Pz))

from a uniform grey background of equal mean luminance. Examples of thepattern reversal and pattern onset VEP are given in Figure 4 and 5, respectively. Amplitude measurements are made between peaks and troughs of the deflections.Peak latency measurements (or implicit time) is taken from the onset of thestimulus to the peak of the component concerned. The flash VEP is elicited by a hand held strobe light. We use flash VEPs to deter-mine if there is any recordable function of the postretinal pathways in childrenwho show hardly any visual response, and to assess misrouting in infants andyoung children.25

The pattern reversal VEP consists of a negative wave around 75 ms, a positivehigh-amplitude component around 100 ms, and a negative of around 145 ms (seeFigure 4). The positive component (P100) is by far the most consistent and usually of highest amplitude and shows remarkable little variation in latencybetween or within individuals. For this reason, we use pattern reversal stimulationmainly to detect a possible abnormality of the conduction velocity of thepostretinal pathways. The normal monocular latency value for our department is107 ± 3 ms (N=32).The waveform of the pattern onset VEP changes until puberty.21, 26 The mature

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Figure 4. Normal pattern reversal VEP

waveform is composed of three main components, CI (predominantly positive,peak latency at approximately 110 ms), CII (negative, approximately 125 ms), andCIII (positive approximately 150 ms) (see Figure 5). CII is of mixed striate andextrastriate origin;27 as check sizes become smaller, the striate component becomesmore prominent, while the extrastriate component diminishes in amplitude.Because the pattern onset VEP is dominated by activity from the central 5º, it maybe used to evaluate macular function.

We also use pattern onset VEPs to assess visual acuity, for instance in preverbal ormentally impaired children, and in children suspected of non-organic visual loss.In acuity VEPs, different check sizes are presented in a series, in which the largerand smaller check sizes are presented sequentially and averaging is carried out oversignal segments covering the full series28 (Figure 5).

To determine misrouting in older children, we analyze the symmetry of thedistribution of pattern onset VEPs with monocular stimulation (see Chapter 2,Figure 5). Furthermore, we record half field responses, obtained simultaneouslyby asynchronous stimulation of the half fields (Figure 6). The potential distribu-tions of striate activity to both half field stimulations will show considerableoverlap and will therefore be difficult to separate. For that reason, relatively coarse

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Figure 5. Normal pattern onset VEP. Different check sizes (from 15’ to 4’) were presented in a series.

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Figure 6. Halffield responses of a normal subject (top) and a patient with misrouting (bottom). In the normalsubject, the differential electrodes O1 – O2 and T5 – T6 show a negative peak with stimulation of the left halffieldsof both eyes, indicating that the responses are found in the right hemisphere. With stimulation of the right half-fields, the response is found in the left hemisphere. In the patient with misrouting, both halffield responses of theleft eye are found in the left hemisphere, of the right eye in the right hemisphere.

checks (60’) are used, to enhance the extrastriate contribution which originatesfrom parts of the brain that are physically much further apart.

From the full field responses (and the flash responses in very young children) wedetermine the chiasmal coefficient.29, 30 The chiasmal coefficient is calculated fromthe differences of the recorded signals from the right and left hemispheres. Bydefinition, it has a value between -1 and +1; it is negative in misrouting, andpositive in normal subjects. The normative criterion value for our laboratory is M-2SD = -0.17.

1.3 Electrophysiology at Bartiméus

The Bartiméus Institute was founded in 1916 to "provide for a Protestanteducation for the blind". The first board of governors consisted only of ministers.In 1926, the first ophthalmologist was appointed, in 1950 the first psychologist.From that time, not only blind but also partially sighted children were admitted.Originally, Bartiméus only took care of children attending special schools for thevisually impaired. Nowadays the greater majority of people referred to Bartiméusfor rehabilitation are adults, mostly elderly people. In 2004 rehabilitation serviceswere given to a total of 14 151 persons; 75% visually impaired adults, 7.5% (1048)children, and 17.5% visually as well as mentally impaired adults.At present, most visually impaired children live at home and visit their localschool, where they are supported in their education and daily life by advisorstrained at Bartiméus.

In 1975 an additional centre for multiple-handicapped children was opened. Thiscentre contained a department for EEG and visual electrophysiology measure-ments. Originally, electrophysiological procedures were mainly performed toassess visual acuity in severely mentally impaired children. The introduction ofpreferential looking tests31 resulted in a reduction of the amount of acuity VEPs,and gradually the use of electrophysiology for diagnostic purposes increased. Prof.J.W. Delleman, who worked at Bartiméus for more than thirty years, especiallyemphasized the importance of assessing the exact medical diagnosis of rare oculardisorders.

In 1998, Bartiméus decided to modernize the electrophysiology department tomake it especially suited for children and mentally disabled people. From thattime, referrals for electrophysiological investigations increased, and at present

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children come from all over the country. In 2005 a total of 152 electrophysio-logical procedures were performed. The electrophysiology department is staffed with three physicists and oneophthalmologist; examinations are performed every Thursday. Four orthoptistsassist at the procedures. Because almost every referred child is visually impaired,we (the ophthalmologist and the physicists) frequently diagnose rare disorders. Wealways judge the electrophysiological findings together, which regularly leads todiscussions about unusual findings, and consequently to suggestions for furtherinvestigations. In 2004, the Bartiméus management provided funds for electro-physiological research, which ultimately resulted in this thesis.

1.4 Aim of the thesis

The aim of the thesis is to demonstrate the value of specialized electrophysiologyin visually impaired children. In Chapter 2, electrophysiological examinations of a three year period areevaluated to assess whether the methods and equipment are suitable for the use inchildren, to report on the diagnostic results, and to describe possible effects onrehabilitation.Chapter 3 reports on the decisive importance of electrophysiological results indiagnosing TRMA (thiamine-responsive megaloblastic anemia). TRMA is asystemic condition that, untreated, may prove fatal. Chapter 4 describes the outcome of ophthalmological and electrophysiologicalexaminations in patients with Rubinstein-Taybi syndrome, a mental retardation-multiple congenital abnormalities syndrome. The study describes the frequency ofthe ophthalmological abnormalities and the consequences with regard to progno-sis and daily life.In Chapter 5 we investigate two children with a very rare disorder: the EEM(ectrodactyly, ectodermal dysplasia, and macular dystrophy) syndrome. These arethe first patients to have been examined in infancy. The results provide insight inthe pathogenesis and prognosis of the ophthalmological abnormalities of thesyndrome.Chapter 6 describes the electrophysiological and clinical characteristics of threepatients with foveal hypoplasia and misrouting. Based on these findings, wequestion widely accepted ideas on albinism and misrouting; we furthermorepropose an alternative theory on the development of foveal hypoplasia.Chapter 7 describes the electrophysiological characteristics of autosomalrecessively inherited congenital stationary night blindness. Apart from the

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standard ERG, we investigated the patients with 15 Hz scotopic flickerstimulation. We report on the possibility to discriminate different forms ofcongenital stationary night blindness with this method. In some of the patients anew mutation was found. The relationship between ERG and DNA analysisreflect on theories on retinal signalling. A summary is given in Chapter 8 together with some thoughts on future research.

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References

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problems. Tenth revision. Vol 1. Geneva: WHO, 1992:457.

2. Rosenberg T, Flage T, Hansen E, Riise R, Rudanko SL, Viggosson G, Tornqvist K. Incidence of

registered visual impairment in the Nordic child population. Br J Ophthalmol 1996;80:49-53.

3. Riise R. Nordic registers of visually impaired children. Scand J Soc Med 1993;21:66-68.

4. Flanagan NM, Jackson AJ, Hill AE. Visual impairment in childhood: insights from a

community-based survey. Child Care, Health Dev 2003;29:493-499.

5. Kocur I, Resnikoff S. Visual impairment and blindness in Europe and their prevention. Br J

Ophthalmol 2002;86:716-722.

6. Rahi JS, Cable N. Severe visual impairment and blindness in children in the UK. Lancet

2003;362:1359-1365.

7. Stewart-Brown SL, Haslum MN. Partial sight and blindness in children of the 1970 birth cohort

at 10 years of age. J Epidemiol Community Health 1988;42:17-23.

8. Loewer-Sieger DH. The prevalence of severe visual impairment in children in the Netherlands.

Child Care Health Dev 1975;1:275-278.

9. Blohmé J, Tornqvist K. Visually impairment in Swedish children. III. Diagnoses. Acta

Ophthalmol Scand 1997:75:681-687.

10. Van der Pol BAE. Causes of visual impairment in children. Doc Ophthalmol 1986;61:223-228.

11. Jacobson KJ, Dutton GN. Periventricular leukomalacia: an important cause of visual and ocular

motility dysfunction in children. Surv Ophthalmol 2000;45:1-10.

12. Dutton GN, Jacobson LK. Cerebral visual impairment in children. Semin Neonatol 2001;6:477-

485

13. Fulton AB, Hartmann EE, Hansen RM. Electrophysiologic testing techniques for children. Doc

Ophthalmol 1989 Apr;71:341-54.

14. Marmor MF, Zrenner E. Standard for clinical electroretinography (1994 update). Documenta

Ophthalmol 1994;89:199-210.

15. Marmor FM, Holder GE, Seeliger MW, Yamamoto S. Standard for clinical electroretinography

(2004 update). Doc Ophthalmol 2004;108:107-114.

16. Yanagida T, Koshimizu M, Kawasaki K, Yonemura D. Microelectrode depth study of the

electroretinographic oscillatory potentials in the frog retina. Doc Ophthalmol 1987;67:355-61.

17. Dawson WW, Trick GL, Litzkow CA. Improved electrode for electroretinography. Invest

Ophthalmol Vis Sci 1979;18:988-91.

18. Miyake Y, Horiguchi M, Ota I, Shiroyama N. Characteristic ERG-flicker anomaly in incomplete

congenital stationary night blindness. Invest Ophthalmol Vis Sci 1987;28:1816-1823.

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19. Fulton AB, Hansen RM. Electroretinography: application to clinical studies of infants. J Pediatr

Ophthalmol Strabismus 1985 Nov-Dec;22:251-5.

20. Fulton AB, Hansen RM, Westall C. Development of ERG responses: the ISCEV rod, maximal

and cone responses in normal subjects. Doc Ophthalmol 2003;107:235-241.

21. Brecelj J. From immature to mature pattern ERG and VEP. Doc Ophthalmol 2003:107:215-224.

22. Westall CA, Panton CM, Levin AV. Time courses for maturation of electroretinogram responses

from infancy to adulthood. Doc Ophthalmol 1999;96:355-379.

23. Hansen RM, Fulton AB. Development of the cone ERG in infants. Invest Ophthalmol Vis Sci

2005;46:3458-62.

24. Manahilov V, Riemslag FC, Spekreijse H. The Laplacian analysis of the pattern onset response

in man. Electroencephalogr Clin Neurophysiol 1992;82:220-224.

25. Apkarian P, Reits D, Spekreijse H. Component specificity in albino VEP asymmetry: maturation

of the visual pathway anomaly. Exp Brain Res 1984; 53:285-294.

26. Spekreijse H. Maturation of contrast EPs and the development of visual resolution. Arch Ital

Biol 1978;116:358-369.

27. Maier J, Dagnelie G, Spekreijse H, van Dijk BW. Principal components analysis for source

localization of VEPs in man. Vision Res 1987;27:165-77.

28. Spekreijse H, Riemslag FCC. Gross potential recording methods in ophthalmology. In: Vision

research, ed. Carpenter RHS, Robson JG; Oxford University Press 1999;8:216-233.

29. Jansonius NM, van der Vliet TM, Cornelissen FW, Pott JW, Kooijman AC. A girl without

chiasm: electrophysiologic and MRI evidence for the absence of crossing optic nerve fibers in a

girl with a congenital nystagmus. J Neuroophthalmol 2001;21:26-29.

30. Pott JWR, Jansonius NM, Kooijman AC. Chiasmal coefficient of flash and pattern visual evoked

potentials for detection of chiasmal misrouting in albinism. Documenta Ophthalmol

2003;106:137-143.

31. Teller DY, Morse R, Borton R, Regal D. Visual acuity for vertical and diagonal gratings in

human infants. Vision Res 1974;14:1433-1439.

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Chapter 2

The key role of electrophysiology in the diagnosis of visually impaired children.

van Genderen MM1, Riemslag FCC 1, Jorritsma FF 1, Hoeben FP 1, Meire FM 2, Stilma JS 3

Accepted for publication in Acta Ophthalmologica Scandinavica

1 Bartiméus Institute for the Visually Impaired, Zeist, The Netherlands2 University Children’s Hospital Koningin Fabiola, Brussel en University Hospital Gent, Belgium3 Department of Ophthalmology, University Medical Centre Utrecht, The Netherlands



The key role of electrophysiology in the diagnosis of

visually impaired children

Abstract

Purpose: To describe the outcome of specialized electrophysiology in visuallyimpaired children.

Methods: Retrospective evaluation of 340 electrophysiological examinationsperformed in 298 children over a three year period (2001-2003),with regard to demographic data, referral pattern, degree of compliance, and diagnostic results. Electrophysiology was per-formed without sedation or anaesthesia. In ERGs, DTL electrodeswere used in combination with on line selection of responses. VEPtesting was performed with seven active occipital electrodes.

Results: Mean age was 7 ± 5 yrs; 72 (24%) of the children were mentally aswell as visually impaired. Main reasons for referral were suspectedposterior segment disease, abnormal visual development, unex-plained low vision, high myopia, and suspected albinism.Compliance was good in 302/340 (88%), partial in 24/340 (7%),and absent in 14/340 (4%) of the examinations. Of the 326 successful procedures, 215 (66%) showed abnormalresults. Tapetoretinal dystrophy (22%), opticopathy (16%), conge-nital stationary night blindness (13%), and cone dystrophy (11%),were the most frequently established diagnoses. Albinism was confirmed in 14 of 24 suspected patients; additionally, unsuspected misrouting was found in 6. In 26 (9%) of the patients, a previouslyestablished diagnosis was changed.

Conclusions: In a specialized setting, electrophysiological examinations cansuccessfully be performed in visually impaired children. The resultsare essential for the final ophthalmological diagnosis and haveimportant consequences for rehabilitation.

Key words: ERG, VEP, visually impaired children, childhood blindness, DTLelectrodes, misrouting

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Introduction

Knowledge of an exact ophthalmological diagnosis is essential in the rehabilitationof a visually impaired child. The diagnosis clarifies the aetiology and consequentlythe genetics of the disorder, it provides an explanation for various visual problemsof the child, and it implies the prognosis. The diagnosis may contribute todecisions about reading print or Braille, or which forms of education or career arefeasible. In several posterior segment disorders visual electrophysiology plays a decisivediagnostic role, because psychophysical testing in infants and young children islimited, and ophthalmological examination may initially reveal a normal lookingretina even in the case of severe retinal dystrophy.1 In older children, funduscopymay be hampered by photophobia or severe nystagmus, or the clinical signs andretinal appearance may fit a number of diagnoses. We report on the outcome of340 electrophysiological examinations in children from a three year period (2001-2003), performed without anaesthesia or sedation, with the use of specializedrecording methods. The outcome parameters were the demographic data of thechildren, the referral pattern, the compliance with the procedure, the type andresult of the electrophysiological examination, and the contribution towards thefinal diagnosis.

Patients and methods

The Bartiméus Institute is one of the Dutch institutes for the rehabilitation ofvisually impaired people, and the only one with an electrophysiology department.In the years 2001, 2002, and 2003, a total of 357 patients were referred forelectrophysiological investigations by ophthalmologists, paediatricians, neuro-logists and genetic specialists; 298 of these patients were children under 18. The 59adult patients were mainly family members of visually impaired children, in whichelectrophysiological results were needed for genetic counselling; the outcome oftheir examinations will not be discussed in this study.

Information regarding the demographic data of the children, the reasons forreferral, and the referring clinicians were obtained from the medical records.

The compliance with the electrophysiological procedure was graded as good if achild could undergo a complete protocol, including, in ERGs, the required 20minutes of dark adaptation. We considered compliance to be partial if the proce-dure had to be shortened, for instance because the child was afraid of the dark.

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Absent compliance meant the procedure had to be aborted before usablemeasurements could be made. Compliance was optimized by the followingmeasures. Small children sat on a parent’s lap while the electrodes were attachedand during the recording. Compliance in infants could sometimes be increased bybottle feeding during the procedure. An assistant was always present in the sameroom as the parents and child to intervene if something went wrong, to giveinstructions, and to reassure and distract the child (and the parents).Communication of the investigator with the assistant, parents, and child remainedpossible because the examination and the recording room were separated only bycurtains. Throughout ERG as well as VEP recordings the assistant encouragedthe child to fixate on the stimuli; the assistant was guided by feedback from theexaminer, who monitored fixation with closed-circuit (infrared) television. DuringVEP stimulation, the assistant drew the child’s attention to the pattern stimulus bymoving noisy toys along the upper part of the screen; the assistant could manuallyhalt averaging if fixation was inadequate.

Visual electrophysiology was performed without sedation or anaesthesia. For the ERG, we used DTL (Dawson, Trick, Litzkow)2 electrodes in all childrenexcept for nine infants (< 1 year of age), who were examined with contact lenselectrodes. The investigator assessed on line every single flash ERG response,excluding from averaging responses that were unreliable because of inadequatefixation, blinking etc. ERGs were recorded according to ISCEV standards.3 Forthe standard ISCEV ERG measurements, Xenon tube flashes (duration ca 10 µs)were delivered in a custom made Ganzfeld dome, at one flash every 2 seconds forthe low (-2.6 ND), and one flash every 5 seconds for the standard ISCEV intensity(mixed response). Subjects were then light adapted for 10 minutes by exposure toa white 30 cd/m2 rod saturating background, and photopic ERGs were recordedto standard ISCEV intensity and to white 30 flashes per second. To the standardISCEV intensities, we added an extended series of stimuli in the dark adaptedcondition: -2.0, -1.6, -1.0, -0.6, -0.3, and +0.6 log; this extended series providesinformation about the consistency of the recorded responses. In the light adaptedcondition we added +0.6 log intensity to the standard ISCEV intensity. In cases ofsuspected congenital stationary night blindness (CSNB) a "Miyake protocol" wasperformed: 30 Hz photopic measurements every minute for ten minutes afterturning on the background illumination. With this protocol the complete andincomplete forms of CSNB (CSNB1 and 2) can be distinguished.4

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VEP testing was performed with seven active occipital electrodes (see Figure 1).Two differential recordings from active electrodes at both hemispheres enhancedand visualised online the hemispheric differences. Additionally, a Laplacianderivation over the striate cortex amplified the contribution of the striate sourcerelative to the contribution of the extra striate source,5 which enhances the signalto noise ratio extensively. Responses were recorded to pattern reversal, patternonset and flash stimulation (flash stimulus energy 1 Joule, duration less than 10 ms;pattern stimulation: VGA monitor, mean luminance 50 cd/m2). We used patternreversal stimulation mainly to detect a possible abnormality of the latency in apatient. (see Figure 2). We assessed misrouting with flash VEPs in infants andtoddlers, pattern onset VEPs in older children.6 Pattern onset VEPs were alsoused to estimate visual acuity, for instance in preverbal or mentally impairedchildren, and in children suspected of non-organic visual loss. Check sizethreshold levels were estimated by presenting the stimuli in a series, in which thelarger and smaller checks were presented sequentially and the averaging wascarried out over signal segments covering the full series.7

Only one EOG was recorded during the three year period, because most disorderswith a reduced EOG also show ERG abnormalities that can be recorded at a muchyounger age. The EOG is essential in the diagnosis of Best’s disease, but children

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Figure 1. Schematic representation of theposition of the electrodes usedfor VEP. Seven active occipitalelectrodes are referenced to afrontal midline electrode. Theactive midline electrode isplaced 2 cm above the inion,with two lateral activeelectrodes on each side, at aspacing of 3 cm. Theseelectrode positions areequivalent to T5, O1, Oz, O2,and T6 of the international 10-20 system. Two differentialrecordings O1 – O2 and T5 – T6

were calculated. Two verticalactive electrodes, also at aspacing of 3 cm, were used forcalculation of the Laplacianderivation.

with this disorder are hardly ever referred to our institute, because the majoritydoes not have significant visual impairment.8

All electrophysiological results were judged by a team of clinical physicists and anophthalmologist. Mean values and standard deviations of normal ERG responseswere obtained from 51 subjects (mean age 10 ± 4 yrs) (Table 1). Because of askewed distribution, mean and standard deviations were calculated afterlogarithmic transformation.9 Responses were considered to be abnormal if ampli-tudes were more than 2SD below the mean, and/or latencies more than 2SD abovethe mean. VEP latencies were determined from the pattern reversal responses, andconsidered abnormal if ≥ 2 SD above the mean (normal monocular value for ourdepartment: 107 ± 3 ms (N=32)).ERG and VEP responses of infants were interpreted according to guidelinesprovided in earlier studies.10-12 The study of Fulton et al 10 provided expected limitsof normal values of the ISCEV rod, maximal, and cone responses in the first yearof life. For instance, a quarter of the normal infants does not have a detectable rodresponse until 5 weeks of age. In evaluating the responses of infants and youngchildren, we took these maturation effects into account. All diagnoses wererecorded before and after the electrophysiological examination.

31


Figure 2. Pattern reversal VEP of anormal subject. Thepositive component (P100)shows remarkably littlevariation in latency betweenor within individuals. Thenormal monocular value forour department is 107 ± 3ms (N=32).

Results

Demographic data of the children340 examinations were performed in 298 children; 43 children had both an ERGand a VEP. An EOG was measured in only one child. 172 children (58%) wereboys, 126 (42%) girls. 72 children (24%) were mentally impaired, and three hadhearing loss. Mean age of the children was 7 ± 5 yrs; the age distribution is shownin Figure 3. 51 children (17%) had a visual acuity below 0.05, 156 (53%) between 0.05 and 0.3,or below the normal range for their age.13, 14 90 children (30%) had a visual acuityof more than 0.3 or within normal range for their age. They were referred becauseof other visual symptoms, for instance night blindness, photophobia, or highmyopia.

Referral patternsMost electrophysiology referrals came from the Bartiméus ophthalmologists, aspart of the diagnostic process and subsequent rehabilitation. Ophthalmologistsfrom other centres for visually impaired children, or from hospitals throughoutthe country, requested 20% (69/340) of the total number of procedures. In 11cases, geneticists or paediatricians asked for electrophysiological investigations.

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stimulus intensity a trough to b peak amplitude (µV) (log to the standard) mean mean – 2 SD

*scotopic -2.6 ND 143 45-2.0 ND 249 98-1.6 ND 312 124-1.0 ND 394 187-0.6 ND 373 193-0.3 ND 418 207

*mixed 0.0 ND 481 243+0.6 ND 486 253

*photopic 0.0 ND 98 36+ 0.6 ND 187 77

* 30 Hz 74 35

Table 1. B-wave amplitudes determined in 51 normal subjects, mean age 10 ± 4 years. *: standard ISCEV ERGmeasurements.

The most frequent reason for referral was suspicion of a retinal disorder. In 85children (90 procedures), this suspicion was based on a combination of symptoms,for instance nystagmus, photophobia, and high hypermetropia. Another 50children (53 procedures) had one presenting symptom only: either nystagmus, orhigh myopia, or night blindness. The second largest group (49 procedures)consisted of children with unexplained low visual acuity, or with a visual acuity thatcould not reliably be determined by psychophysical tests. This group consistedmainly of severely mentally impaired children, and children suspected of non-organic visual loss. Other important reasons for referral were probable albinism,and infants < 1 yr of age with abnormal visual development, for instance, absentfixation and following, or roving eye movements.

ComplianceGood compliance was achieved in 302/340 procedures (88%). Compliance waspartial in 24 (7%) procedures, but in all these cases a diagnosis could beestablished. Fourteen procedures (4%, 11 ERGs and 3 VEPs) had to be abortedbecause of non-cooperation; ten involved severely mentally impaired children, theremaining four mentally normal children between one and five years of age. Thesechildren had miscellaneous disorders; no diagnosis seemed therefore predominantin the failed procedures. A successful procedure was possible in 62/72 (86%) ofmentally impaired children, and 221/226 (98%) of children with normaldevelopment.

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Figure 3. The age distribution of the electrophysiologically examined children (N=297, 340 procedures)

Electrophysiological resultsThe results of all successful electrophysiological procedures are summarised inTable 2. The table does not show the one EOG, which was normal, and fiveprocedures that yielded inconclusive results, despite good measurements.In 82/90 procedures (91%) performed for suspected retinal disease, anelectrophysiological diagnosis could be established. Examples of the ERGs inseveral diagnoses are shown in Figure 4. The most frequently found retinaldisorders were were progressive tapetoretinal dystrophies (TRD, rod-cone as wellas cone-rod dystrophies) (48), CSNB (29), cone dysfunction/dystrophy (24), andachromatopsia (15). Of the patients with CSNB, eight had CSNB1 ("complete"form), fourteen CSNB2 ("incomplete" form), and seven autosomal recessiveCSNB. Nine patients with unexplained low visual acuity proved to have non-organic visual loss. 34 procedures were performed in 25 infants; nine infants had both an ERG and aVEP. 35% (12/34) of these procedures yielded abnormal results, VEPs being moreoften abnormal than ERGs (10 and 2, respectively). 76% (22/29) of children withhigh myopia as presenting symptom had abnormal electrophysiological findings;cone dystrophy was diagnosed in half of them. Misrouting was demonstrated in 14 of 24 suspected cases; an example is given inFigure 5. Additionally, misrouting was found in six patients not referred for thisdiagnosis. Opticopathy, with or without cerebral visual impairment, was confirmed in 17 ofthe 21 suspected cases, in 17 patients with miscellaneous symptoms, and in sixteenmentally impaired patients referred for visual acuity assessment. Three-quarters of the children with abnormal electrophysiological findings had anophthalmological disorder only, like, for instance CSNB. In the remainingquarter, the electrophysiological diagnosis contributed to the identification of asystemic condition, for example retinal dystrophy as part of a syndrome ormetabolic disease.

Change of diagnosisIn 26 patients the electrophysiological results altered a previously assumed diag-nosis. In 22 of these cases, the definitive diagnosis considerably changed theprognosis of the condition, for instance CSNB instead of TRD (better prognosis)or TRD instead of X-linked myopia (worse prognosis). (Table 3)

34


35


Figure 4. Standard ISCEV ERG responses of a normal subject (top left), a patient with achromatopsia (top right), a patientwith CSNB (bottom left), and a patient with retinal dystrophy (bottom right). The diagnoses in the patients wereconfirmed by DNA analysis. Normal subject: from top to bottom the scotopic responses, mixed response, oscillatory potentials, photopicresponses, and photopic response to 30 Hz flicker. Achromat: after the scotopic and mixed b-wave, a blink artefact can be seen, probably because of photophobia. Still, based on these recordings the diagnosis could be esta-blished. DNA analysis: CNGB3 mutation36 CSNB patient: the scotopic response is absent, the mixed responseshows the characteristic electronegative waveform, the photopic responses are normal. DNA analysis: GRM6mutation.37

Retinal dystrophy: notice the scale difference; there are only very small photopic responses. DNA analysis: RPE65mutation.38

36


Figure 5.Responses to pattern onset-offset stimulation of a normal subject (top), and a patient with misrouting (bot-tom). In the patient with misrouting, the differential recordings O1-O2 and T5-T6 show a positive peak withstimulation of the right eye, which means that the response is found in the left hemisphere. In the left eye, thispeak is negative, caused by a response of the right hemisphere.

37


Reason referral/ presenting ERG VEP ERG VEPsymptom (total no. normal Abnormal abnormal misroutingof procedures)Suspicion of retinal problem 7 1 TRD 36 4*(90) CSNB 17

Achr/blue cone 15/3Cone dystrophy 6 Retinoschisis 1

Unexplained low vision, visual acuity assessment (49) 21 9 TRD 2 16 1Infant** (34) 14 8 Cone dystrophy 2 9 1 High myopia (> -6 D) (29) 7 Cone dystrophy 11 1

CSNB 7TRD 3 1

Albinism (24) 9 1 14Opticopathy/CVI (21) 2 TRD 1 17 1Nystagmus (16) 2 5 Cone dystrophy 5 2

CSNB 2General syndrome (16) 6 3 TRD 5 2 Night blindness (8) 3 CSNB 3 1

TRD 1 1Follow up (25) 1 2 13 9Family screening (8) 5 2 1Total 320 66 41 134 59 20

(33%) (34%) (67%) (49%) (17%)Normal Abnormal

* Opticopathy secondary to TRD; ** Children<1 yr with abnormal visual development, for instance no fixing orfollowing, roving eye movements, or nystagmus; infants with suspected albinism are counted under "albinism".Abbreviations: Achr: achromatopsia, Blue cone: blue cone monochromacy, CSNB: congenital stationary nightblindness, CVI: cerebral visual impairment, TRD: tapetoretinal dystrophy

Table 2. Results of electrophysiological measurements (N=320)

Discussion

With an electrophysiology department especially adapted for children we wereable to establish a diagnosis in 278/298 (94%) of the referred children, without theuse of anaesthetics or sedation. The high success rate may partly be attributed toselection of patients. Most children referred for electrophysiology were firstexamined ophthalmologically, at which time the ophthalmologist could ascertainthe probability of sufficient compliance for the procedure. Also we regularly

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original diagnosis no p/s* diagnosis after no p/s*electrophysiology

TRD 8 p CSNB 3 sachromatopsia 1 sblue cone monochromacy 3 scone dystrophy 1 p

LCA 6 p optic atrophy/CVI 1 sCSNB 3 sachromatopsia 1 scone dystrophy 1 p

cone dystrophy 2 p non-organic visual loss 1 sTRD 1 p

CSNB 4 s myopia 2 scone dystrophy 1 pTRD 1 p

X-linked myopia 1 s TRD 1 poptic atrophy 1 s TRD 1 pnon-organic visual loss 1 s TRD 1 p

pcongenital nystagmus 1 s cone dystrophy 1 palbinism 2 s congenital nystagmus 1 s

cone dystrophy 1 p

*: p: progressive condition; s: stationary conditionAbbreviations: CSNB: congenital stationary night blindness, CVI: cerebral visual impairment, LCA: Leber’scongenital amaurosis, TRD: tapetoretinal dystrophy

Table 3. Change of diagnosis after electrophysiological examination (N=26)

decided to postpone measuring ERGs in toddlers, unless parents or clinicians hada very urgent reason for knowing the diagnosis. Despite this patient bias and thetoddler "dip" in ERGs, as can be seen in Figure 3, more than half of the investi-gated children were less than eight years of age. Technical measures to maximize success rate were using an extended stimulusseries which made it easier to discern possible trends in the responses, the on-linerejection of unreliable ERG responses, and the use of DTL electrodes. On-lineselection of responses was especially advantageous in cases of nystagmus, becausenystagmus may make recordings very noisy. DTL electrodes are far morecomfortable than contact lens electrodes, have negligible risk of corneal abrasions,and yield much larger amplitudes than skin electrodes.15 Furthermore, monitoringfixation in a child with DTL electrodes is much easier than with contact lenselectrodes. Previous studies describe about 30% lower amplitudes with DTLelectrodes compared to contact lens electrodes.16, 17 However, with selection ofresponses and fixation monitoring we were able to obtain amplitudes considerablyhigher than previously described. For instance, we measured a mean (ISCEVstandard) mixed response of 481 µV ± 119 mV (N=51) (Table 1). This is in thesame range as found in adults measured with contact lens electrodes,10 but in ourcase in a much younger and less cooperative population. In VEP testing, fixation was also closely monitored. Furthermore, we used moreactive occipital electrodes than recommended by ISCEV standards, combinedwith a Laplacian derivation. Visually (and sometimes also mentally) impairedchildren frequently have fixation problems or nystagmus.18, 19 The Laplacianderivation enhanced the signal to noise ratio, while the use of more electrodesincreased the chance of a successful recording. An additional advantage proved tobe the detection of misrouting in children not suspected of albinism.Lastly, we tried to optimize compliance by working in a specially equippedenvironment. The continuous presence of an assistant assured immediate inter-vention if something went wrong.

Our study depicts an overall picture of diagnoses found in visually impairedchildren. Previous studies described electrophysiological results of children withone defined abnormality only: for instance children with nystagmus,20, 21 refractiveerrors,22, 23 or cone dysfunction.24

Cerebral visual impairment and optic atrophy are the most frequent causes ofvisual impairment in children in the western world.25-29 Patients with these disor-ders were often already diagnosed with cerebral damage by their paediatricians or

39


child neurologists at the time of referral to our institute. Electrophysiology (VEPs)in these children was therefore mainly used for assessing visual acuity or follow-up. Consequently, the greater part (63%) of procedures in our series consisted ofERGs.In previous studies on the causes of visual impairment in children, retinal disordershave generally been lumped together.25, 26, 30-32 In our study, 120 children proved tohave a hereditary retinal disorder. Progressive conditions (TRD and cone-dystrophy) occurred in 72/120 (60%), stationary disorders (CSNB, achroma-topsia, blue cone monochromacy, and retinoschisis) in 48/120 (40%). Therelatively high incidence of CSNB and cone dystrophy may be due to the fact thatwe examined all highly myopic children that had no obvious reason for theirmyopia, as, for instance, Down’s syndrome. Half of these children had a visualacuity of more than 0.3 (but less than 1.0), and thus were not visually impairedaccording to the WHO definitions.33 Quite often their subnormal visual acuityhad been contributed to the myopia itself. Previous studies also have stressed theimportance of investigating children with high myopia,22, 23 one study showing thatonly 8% of children under ten with myopia of more than 6 diopters did not havean underlying ocular or systemic condition.23

60% of children with reported night blindness proved to have a retinal disorder.On the other hand, night blindness was not the presenting symptom in themajority of children diagnosed with CSNB. Especially children with CSNB2("incomplete" CSNB) often had more problems related to their cone dysfunction,like low visual acuity and photophobia, than problems with night vision.The electrophysiology results changed a previously established diagnosis in 26cases. More than half of these cases (14/26) concerned diagnoses of Leber’scongenital amaurosis (LCA) or early TRD. These diagnoses had all beenelectrophysiologically established in infancy or at a very young age, often underanaesthesia. Earlier studies have shown that ERG amplitudes are significantlylower in very young children compared to those in older children,10, 11 especiallythe scotopic and maximal responses. Also, several anaesthetics may influence ERGoutcomes.34 Furthermore, some cases of CSNB have been described with initiallyvery low ERG amplitudes, which led to the impression of LCA.35

The change of diagnosis had profound implications for the rehabilitation of thechildren. The approach of a child with non-organic visual loss is very muchdifferent from the approach of a child with retinal dystrophy. Several children

40


thought to suffer from night blindness were shown to have a normal ERG andnormal dark adaptation, which made special illumination measures unnecessary.The child that had its diagnosis changed from LCA to achromatopsia waseducated to read print instead of Braille.

Conclusion

In a specialized setting, electrophysiology can reliably be performed in visuallyimpaired children, without anaesthesia or sedation. Because of several technicalmeasures, we were able to obtain considerable ERG amplitudes with DTLelectrodes. In VEP testing, using more active electrodes not only increased thechance of a successful recording, but also led to the detection of unsuspectedmisrouting.

Based on this study, we recommend early referrals in infants (because of theproblems associated with performing ERGs in toddlers), children with unexplain-ed high myopia, and night blindness. Rehabilitation of visually impaired childrenmay be directly influenced by electrophysiological results, especially if they lead toa change in diagnosis.

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References

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4. Miyake Y, Horiguchi M, Ota I, Shiroyama N. Characteristic ERG-flicker anomaly in incomplete

congenital stationary night blindness. Invest Ophthalmol Vis Sci 1987;28:1816-1823.

5. Manahilov V, Riemslag FC, Spekreijse H. The Laplacian analysis of the pattern onset response

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7. Spekreijse H, Riemslag FCC. Gross potential recording methods in ophthalmology. In: Vision

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8. Ponjavic V, Eksandh L, Andreasson S, Sjostrom K, Bakall B, Ingvast S, Wadelius C, Ehinger B.

Clinical expression of Best's vitelliform macular dystrophy in Swedish families with mutations in

the bestrophin gene. Ophthalmic Genet 1999;20:251-7.

9. Altman DG. Skewed distributions. In: Practical statistics for medical research. London,

Chapman & Hall 1997: 60-63.

10. Fulton AB, Hansen RM, Westall CA. Development of ERG responses: the ISCEV rod, maximal

and cone responses in normal subjects. Doc Ophthalmol 2003;107:235-241.

11. Westall CA, Panton CM, Levin AV. Time courses for maturation of electroretinogram responses

from infancy to adulthood. Doc Ophthalmol 1999;96:355-379.

12. Brecelj J. From immature to mature pattern ERG and VEP. Doc Ophthalmol 2003;107:215-224.

13. Teller DY, Morse R, Borton R, Regal D. Visual acuity for vertical and diagonal gratings in

human infants. Vision Res 1974;14:1433-1439.

14. Salomao SR, Ventura DF. Large sample population age norms for visual acuities obtained with

Vistech-Teller Acuity Cards. Invest Ophthalmol Vis Sci 1995; 36:657-70.

15. Kriss A. Skin ERGs: their effectiveness in paediatric visual assessment, confoudning factors, and

comparison with ERGs recorded using various types of corneal electrode. Internat J

Psychophysiol 1994;16:137-146.

16. Hennessy MP, Vaegan. Amplitude scaling relationships of Burian-Allen, gold foil and Dawson,

Trick and Litzkow electrodes. Doc Ophthalmol 1995; 89:235-248.

17. McCullouch DL, Van Boemel GB, Borchert MS. Comparison of contact lens, foil, fiber and skin

electrodes for patterns electroretinograms. Doc Ophthalmol 1998;94:327-340.

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18. Dutton GN, Jacobson LK. Cerebral visual impairment in children. Semin Neonatol 2001;6:477-

485.

19. Jacobson KJ, Dutton GN. Periventricualr leukomalacia: an important cause of visual and ocular

motility dysfunction in children. Surv Ophthalmol 2000;45:1-10.

20. Brecelj J, Stirn-Kranjc B. Visual electrophysiological screening in diagnosing infants with

congenital nystagmus. J Clin Neurophysiol 2004;115:461-470.

21. Lorenz B, Gampe E. Analysis of 180 patients with sensory defect nystagmus (SDN) and

congenital idiopathic nystagmus (CIN). Klin Monatsbl Augenheilkd 2001; 218:3-12.

22. Flitcroft DI, Adams GG, Robson AG, Holder GE. Retinal dysfunction and refractive errors: an

electrophysiological study of children. Br J Ophthalmol 2005;89:484-488.

23. Marr JE, Halliwell-Ewen J, Fisher B, Soler L, Ainsworth JR. Associations of high myopia in

childhood. Eye 2001;15:70-74.

24. Kelly JP, Crognale MA, Weiss AH. ERGs, cone-isolating VEPs and analytical techniques in

children with cone dysfunction syndromes. Doc Ophthalmol 2003;106:289-304.

25. Alagaratnam J, Sharma TK, Lim CS, Fleck BW. A survey of visual impairment in children

attending the Royal Blind School, Edinburgh using the WHO childhood visual impairment

database. Eye 2002;16:557-561.

26. Blohmé J, Tornqvist K. Visually impairment in Swedish children. III. Diagnoses. Acta

Ophthalmol Scand 1997;75:681-687.

27. Hansen RM, Flage T, Rosenberg T, Rudanko SL, Viggosson G, Riise R. Visual impairment in

Nordic children. III. Diagnoses. Acta Ophthalmol (Copenh) 1992;70:597-604.

28. Rahi JS, Cable N. Severe visual impairment and blindness in children in the UK. Lancet 2003;

362:1359-1365.

29. Rosenberg T, Flage T, Hansen E, Riise R, Rudanko SL, Viggosson G, Tornqvist K. Incidence

of registerd visual impairment in the Nordic child population. Br J Ophthalmol 1996;80:49-53.

30. Flanagan NM, Jackson AJ, Hill AE. Visual impairment in childhood: insights from a

community-based survey. Child Care, Health Dev 2003;29:493-499.

31. Kocur I, Resnikoff S. Visual impairment and blindness in Europe and their prevention. Br J

Ophthalmol 2002;86:716-722.

32. Loewer-Sieger DH. The prevalence of severe visual impairment in children in the Netherlands.

Child Care Health Dev 1975;1:275-278.

33. World Health Organization. International statistical classification of diseases and related health

problems. Tenth revision. Vol 1. Geneva: WHO, 1992:457.

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34. Tremblay F, Parkinson JE. Alteration of electroretinographic recordings when performed under

sedation of halogenate anesthesia in a pediatric population. Doc Ophthalmol 2003;107:271-279.

35. Weleber RG, Tongue AC. Congenital stationary night blindness presenting as Leber’s

congenital amaurosis. Arch Ophthalmol 1987;105:360-365.

36. Sundin OH, Yang JM, Li Y, Zhu D, Hurd JN, Mitchell TN, Silva ED, Maumenee IH. Genetic

basis of total colourblindness among the Pingelapese islanders. Nat Genet 200l;25:289-93.

37. Zeitz C / Van Genderen M, Neidhardt J, Luhmann UF, Hoeben F, Forster U, Wycisk K, Matyas

G, Hoyng CB, Riemslag F, Meire F, Cremers FP, Berger W. Mutations in GRM6 cause

autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz

flicker electroretinogram. IOVS 2005;46:4328-35.

38. Yzer S, van den Born LI, Schuil J, Kroes HY, van Genderen MM, Boonstra FN, van den Helm

B, Brunner HG, Koenekoop RK, Cremers FP. A Tyr368His RPE65 founder mutation is

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Chapter 3

Thiamine-responsive megaloblastic anemia syndrome (TRMA) with cone-roddystrophy.

Meire FM 1,2, Van Genderen MM2, Lemmens K3, Ens-Dokkum MH4

Ophthalmic Genet 2000;21:243-50

1 Department of Pediatric Ophthalmology, University Hospital, Gent, Belgium2 Bartiméus Institute for Visually Impaired Children, Zeist, The Netherlands3 University Clinic of Antwerpen, Belgium 4 Institute for the Deaf, Effatha, Zoetermeer, The Netherlands



Thiamine-responsive megaloblastic anemia syndrome

(TRMA) with cone-rod dystrophy.

Abstract

Thiamine-responsive megaloblastic anemia (TRMA) is an autosomal recessivedisease in which the active thiamine uptake into cells is disturbed. The molecular basis underlying the disorder has been related to mutations in thegene SLC19A2 on chromosome 1q23.3 that encodes a functional thiamine trans-porter. The protein is predicted to have 12 transmembrane domains. TRMA ischaracterized by sensorineural deafness, diabetes mellitus, megaloblastic anemia,and cardiomyopathy. Optic nerve atrophy and retinal dystrophy have beenreported in a small number of patients. We report a 15-year-old girl with TRMAand cone-rod dystrophy and confirm that retinal dystrophy may form part of thesyndrome. Differential diagnosis of syndromes with deafness, diabetes mellitus,and optic nerve atrophy or retinal dystrophy are discussed. The authors suggestthat ERG be performed in all patients with TRMA.

Key words

Thiamine-responsive megaloblastic anemia; retinal dystrophy; cone-rod dystro-phy; differential diagnosis; review

Introduction

The first report on thiamine-responsive megaloblastic anemia (TRMA) in an 11-year-old girl was published in 1969 by Rogers et al.1 TRMA is an autosomalrecessive disorder characterized by megaloblastic anemia, diabetes mellitus, andsensorineural deafness. More diffuse myelodysplasia affecting erythrocytes, as wellas granulocytes and thrombocytes, may also occur.2 In addition, some patientsshow congenital heart anomaly3 and/or suffer from a cerebrovascular accident.4 Upnow, the total number of reported families is limited to 25. Because of the patho-genic, therapeutic, and genetic aspects, some patients with TRMA have beendiscussed more than once in the literature. The syndrome has never beendiscussed in the ophthalmologic literature. Thiamine transport across the intestineand into cells proceeds by a co-existent dual system. At physiological concentra-tions, thiamine is transported primarily by an active process by way of a high-affinity transporter. At higher pharmacological concentrations, thiamine uptake is

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predominantly by a passive process.5 A defective high-affinity thiamine transporterhas been associated with TRMA.6 The administration of pharmacological doses ofthiamine controls the anemia in all patients and decreases the insulin requirementsin some.4,7-10 Once developed, sensorineural deafness is unresponsive to thiamine.However, the institution of treatment could possibly prevent further progressionof hearing loss.8

The TRMA syndrome has been mapped to chromosome 1q23.2-23.3.11 Recently,the causal gene was identified as SLC19A2 by Fleming et al.12 and cloned by Diazet al.13 The gene encodes a thiamine transporter predicted to have 12transmembrane domains. Two frameshift mutations have been detected.14 A girlwith cone-rod dystrophy is described in this report. The diagnosis of TRMA wasdelayed until she was 15 years old.

Case report

The propositius [M.B. °10-02-1984 (female)] was one of five children. Thesiblings as well as the parents were healthy. The parents were consanguineous andof Turkish origin. At age 1 year, the child was found to be deaf. She developeddiabetes mellitus and a right spastic hemiplegia after a left cerebral vascularocclusion. At age 7 years, she was admitted with high fever and pancytopenia(erythrocytes: 2,370,000; thrombocytes: 156,000; leukocytes: 4700; eosinophils:4%; reticulocytes: 49%; lymphocytes: 39%; monocytes: 8%; hemoglobin: 4.3mmol/l; hematocrit: 0.22). The peripheral blood smear revealed poikilocytosis,anisocytosis, and anisochromia. The serum levels of vitamin B12 and folic acid werenormal. Investigations failed to demonstrate any infection. Examination of bonemarrow showed normal cellularity. Multiple blood transfusions controlled theanemia. Cardiological examination revealed an ASD type II and cardiomyopathy.During this hospitalization, vision loss was observed for the first time. Her visualacuity was 0.5 in both eyes and the ERG was consistent with a cone-rod dystrophy(Table 1).The scotopic ERG (measured with -2.0 ND of the Standard Flashintensity) had a normal waveshape with an onset latency of 48 ms and a b-wavelatency of 93 ms. The b-wave amplitude was 23 µV (14% of the normal mean).The latency decreased with increasing flash intensity, but less so than normal,suggesting the absence of cone (or cone-mediated) contribution at the higherintensities (above -1.0 ND). Furthermore, the amplitude did not increasesubstantially. The response at standard flash intensity ( mixed rod-cone response)was reduced even more. The waveshape suggests only rods (or rod-mediated)contribution to this response. In the photopic adaptation, the responses showed

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prolonged latencies, and amplitudes less than 10 µV. The waveshape suggests rod(mediated) responses only. The photopic 30 Hz stimulation did not producerecognizable responses. On repetition, both in the dark- and the light-adaptedcondition, responses tended to decrease even more. In conclusion, the ERGshowed grossly reduced normal latency scotopic rod-mediated responses withabsent cone responses. The co-existence of a hematologic disorder, cardiomyo-pathy, and retinal disorders was suggestive of an underlying mitochondrial disaese.A heart biopsy did not support this hypothesis as there were no characteristics ofa mitochondrial disorder. The biopsy showed a ventricle dysplasia. At the age of15 years, the parents visited the Bartiméus Institute for Visually Impaired Children

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Figure. 1. (A) The posterior pole of the right eye of the patient with TRMA at age 15 years. Notice the bull’seye maculopathy. (B) The peripheral part of the fundus of the patient shows bone spicules.

Intensity (ND) Lat onset (a) ms Latency b ms Amp b-on (a) uV

-2.0 45/51 93/93 24/22 -1.6 31/44 90/90 25/27 -1.0 26/30 71/71 33/36 -0.6 24/29 70/70 34/42 0.0 (stand flash) 35/35 (?) 57/57 10/12 Repeat 35/35 56/56 4/6 Fotopic 0.0 35/35 56/56 7/7 Repeat 35/37 53/50 5/5 30 Hz absent

University Clinic of Leiden.

Table 1. ERG of our patient at age 10 years.

as their child had become deaf-blind. Diabetes was controlled with insulin andpancytopenia with multiple blood transfusions (up to 6 times a year).Ophthalmologic examination revealed a visual acuity of 0.1 in both eyes.Fundoscopy showed bull’s eye maculopathy with peripheral pigmentary changes(Fig. 1). The possible diagnosis of TRMA was made and thiamine 100 mg/daynormalized the blood formule (Fig. 2). The diabetes mellitus had still notimproved.

Discussion

Transfusion-dependent patients often need the administration of intraveneousdesferrioxamine. Desferrioxamine is a chelating agent used in the management ofiron overload conditions such as transfusion-related hemosiderosis. Resultant

50


Figure. 2. Hemoglobin levels before and after thiamine administration.

toxic pigmentary retinal degeneration may appear within a few months and maybe progressive.15

Our patient did not undergo such treatment and therefore the hypothesis of a toxicretinopathy can be rejected. The association of sensorineural deafness, diabetesmellitus, and visual impairment requires the differential diagnosis betweenfollowing sydromes: TRMA, Pearson, DIDMOAD, Alström, and hydroxyacyl-

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TRMA DIDMOAD Alström Pearson Hydroxyacyl-CoA !↓ Dehydrogenase Kearns Sayre deficiency

Deafness Onset: <1y Onset: 10y-20y Onset: 10y-20y Adult onset Onset: <1y Progressive mild mild mild (KS) severe

Diabetes mellitus Early onset Onset: 10y-20y Onset: 10y-20y Early onset: <10y Early onsetInsulin- insulin- insulin- insulin- insulin-dependent dependent resistant dependent dependent

Retinal dystrophy Onset: >5y - Onset: <1y Early onset (P) Rare severe adult onset (KS) Early onset

Optic nerve atrophy Early onset Onset: 5y-10y - - - Rare

Anemia Onset: <1y - - Early onset (P) -Cardiomyopathy Onset: <1y - Acute: <1y Late onset (KS) Early onset

(late onset) sudden death

Diabetes insipidus - Onset: 10y-20y - - Myopathy - - - Adult onset (KS) Early onset

severe Inheritance AR AR AR mtDNA deletion AR

1q23.2 4p16.1 2p14-p13 2p23 SLC 19 A2 WFS1 genegene (thiamine (membrane protein)transporter)

AR, autosomal recessive.

Table 2. Syndromes with deafness, diabetes mellitus, optic atrophy, or retinal dystrophy.

CoA dehydrogenase deficiency. The clinical features, age at onset of the symptoms,and genetic aspects of these syndromes are discussed in Table 2. The clinical pictureof TRMA is related to the uptake and the metabolism of thiamine.

Thiamine (vitamin b1) uptake and metabolism

All experiments show that thiamine uptake follows a biphasic curve, nonlinear atlow concentrations and linear at high concentrations. This indicates that twodifferent systems are involved in thiamine transport. At low thiamine concentra-tions (<2 mmol/l), transport proceeds by a saturable, high affinity/low capacitycarrier. This is a specific active process, requiring energy. At high concentrations(>2 mmol/l), thiamine diffuses passively by a low affinity/high capacity mechanismthat is nonsaturable. When food is the only source, thiamine concentrations in theintestinal bow are <2 mmol/l and uptake proceeds mainly by the activemechanism. The mucosa of the duodenum has the highest rate of thiamineuptake.5 Thiamine transport is basically similar in the plasma membranes ofdifferent cells and across the blood-brain barrier. Intracellular thiamine isconverted by thiamine pyrophosphokinase (TPK) to thiamine pyrophosphate(TPP)(Fig. 3). TPP serves as a cofactor. The complex metabolic pathways shownin Figure 3 suggest that thiamine deprivation can lead to dysfunction of the Krebscycle, shutting down mitochondrial energy production. Mitochondrial changes, inturn, may play an important role in activation of cell apoptosis.6

Thiamine-responsive megaloblastic anemia syndrome (TRMA)

TRMA patients lack the saturable, active uptake mechanism, while thenonsaturable passive component is present. So, at physiological concentrations(food as the only source), thiamine is not transported normally and vitamindeficiency occurs. TRMA patients also have reduced TPK activity, furtherinterfering with normal vitamin accumulation in the cells. As thiamine transportis basically similar in all cells, it is most likely that thiamine uptake is defective inall cells of TRMA patients. The defect results in a state of vitamin deficiencycausing metabolic aberrations in various tissues, including auditory pathways, thebrainstem, the pancreas, and the hematopoietic system. Deafness, diabetesmellitus, anemia, cardiac abnormalities, and optic atrophy can be induced by adisturbance of thiamine-dependent biochemical processes.7,9 A variety of anemiascan be found in TRMA: e.g., megaloblastic, sideroblastic, or aplastic, all of whichrespond to therapy. So, thiamine may play a role not only in DNA metabolism(megaloblastic changes) and heme synthesis (ring sideroblastic changes), but also

52


Table 3. Families with TRMA andoptic atrophy or retinaldystrophy.

( ) References that reportthe same family. nm, notmentioned; abn, abnormal;ERG*, ‘electroretinographic findings before and aftertherapy did not differ’: theauthors reported optic atrophy in their patient, although this remark is suggestive of retinal dystro-phy. optic atrophy or retinaldystrophy.

Figure. 3. Thiamine is converted by TPK to TPP.TPP is an important co-factor in different metabolic pathways. Thiamine deprivation mayinfluence mitochondrial energy production.

Optic atrophy Poggi et al. (1984) 8 (7,9,16,20) ERG nm Schwingshandl & Borkenstien (1989)18 ERG* Vora & Lilleym (1993)21 (14,22: fam 8) ERG nm Raz et al. (1998)22 (fam 5) ERG nm

Retinal dystrophy Cagianut et al. (1977)19 ERG nm

Borgna-Pignatti et al. (1989)17 (11,14,22: fam2) ERG abn Grill et al. (1991)23 ERG abn Morimoto et al. (1992)24 (14,22: fam10) ERG nm Meire et al. (2000) ERG abn

53


in the regulation of hematopoiesis at the stem-cell level.2 The activity of TPP-dependent enzymes is not the same in every tissue and also shows interindividualdifferences. Co-factor depletion slows the enzymatic reactions. The slower thepre-existent enzymatic activity, the more the individual or the tissue is sensitive tothiamine deficiency. This explains why different tissues are differently affected.Particular neuron regions such as the brainstem show an increased sensitivity tolow thiamine levels, resulting in slowly progressive and irreversible lesions. Acutepathological changes in the central nervous system only arise when total thiaminecontent in the brain falls below 20% of normal levels. As TRMA patients still havesome thiamine uptake, most CNS structures maintain sufficient levels to preventsevere neurological manifestations as in acute avitaminosis.16

Since TRMA patients retain their nonsaturable diffusive transport mechanism,pharmacological concentrations of thiamine (>2 mmol/l) can still be taken up.Anemia and to some degree diabetes are reversible, while hearing loss isirreversible.4,7-10 Bone marrow seems less sensitive to deprivation. As long ashematopoietic stem cells are still present, the erythroid lineage can repopulate.The persistent monocytosis shows that the progenitors remain sensitive tothiamine. Diabetes in TRMA is variably reversible, probably because either the b-cells of the pancreas or the target tissue have intermediate sensitivity to cellularthiamine deficiency. Neurons have high energy requirements, and thiaminedeficiency can therefore lead more easily to cell death, resulting in optic nerveatrophy and progressive deafness.6

Obligate heterozygotes and a patient’s relatives often show some clinical featurestypical of manifest TRMA, including diabetes, decreased glucose tolerance, ordeafness.16

Ocular symptoms are inconsistent in association with TRMA. Optic nerve atrophyand retinal dystrophy have been reported. In the earlier literature, some TRMApatients have been diagnosed with either DIDMOAD17,18 or Alstrom syndrome.19

Families with TRMA and optic atrophy or retinal dystrophy are summarized inTable 3. Most authors who mentioned visual impairment did not performelectrophysiologic investigations. Therefore, it is unclear if the optic atrophy intheir patients was secondary to a retinal dystrophy. Follow-up of the patients,initially reported by Poggi et al,8 revealed progressive optic nerve subatrophy.7 Itseems likely that optic nerve atrophy or retinal dystrophy are observed in olderuntreated children in whom the diagnosis was not made previously. We report a child with TRMA and cone-rod dystrophy. The diagnosis in our

54


patient was made only at age 15 years; therefore, from the ophthalmological pointof view, it may not be possible to evaluate the role of early treatment in theprevention of cone-rod dystrophy. As intracellular thiamine deprivation leads to a dysfunction of the Krebs cycle andtherefore to dysfunction of mitochondrial energy production, it is also quitepossible that some individuals are more sensitive to develop apoptotic cell loss intheir retinal ganglion cells and retinal photoreceptors. Especially individuals withTRMA, who carry the heterozygote state of a hereditary dystrophy, could be moresensitive to develop ocular symptoms. To clarify the prevalence and the evolutionof ocular disease in TRMA, we suggest that ocular examination and ERG beperformed in all patients.

55


References

1. Rogers L, Porter F, Sidbury J. Thiamine-responsive megaloblastic anemia. J Pediatr

1969;74:494-504.

2. Bazarbachi A, Muakkit S, Ayas M, Taher A, Salem Z, Solh H, Haidar J. Thiamine-responsive

myelodysplasia. Br J Haematol 1998;102(4):10981100.

3. Abloud M, Alexander D, Najjar S. Diabetes mellitus, thiamine-dependent megaloblastic anemia

and sensorineural deafness associated with defective a-ketoglutarate dehydrogenase activity.

J Pediatr 195; 107:537-541.

4. Mandel H, Berant M, Hazani A, Naveh Y. Thiamine-dependent beriberi in the ‘thiamine-

responsive anemia syndrome’. N Engl J Med 1984;311:836-838.

5. Hoyumpa A, Strickland R, Sheehan JJ, Yarborough G, Nichols S. Dual system of intestinal

thiamine transport in humans. J Lab Clin Med 1982; 99(5):701-708.

6. Stagg AR, Fleming JC, Baker MA, Sakamoto M, Cohen N, Neufeld EJ. Defective high-affinity

thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome

fibroblasts. J Clin Invest 1999;103(5):723-729.

7. Rindi G, Casirola D, Poggi V, De Vizia B, Patrini C, Laforenza U. Thiamine transport by

erythrocytes and ghosts in thiamine-responsive megaloblastic anaemia. J Inherit Metab Dis

1992;15(2):231-242.

8. Poggi V, Longo G, DeVizia B, Andria G. Thiamine-responsive megaloblastic anaemia: a

disorder of thiamine transport? J Inherit Metab Dis 1984; 77(2):153-154.

9. Rindi G, Patrini C, Laforenza U, Mandel H, Berant M, Viana MB, Poggi V, Zarra AN. Further

studies on erythrocyte thiamine transport and phosphorylation in seven patients with thiamine-

responsive megaloblastic anaemia. J Inherit Metab Dis 1994; 17(6):667-677.

10. Schorderet D, Hirt L, Masseret E, De Crousar H, Theintz G. Diabetes mellitus, sensorineural

deafness and megaloblastic anemia: a case of Rogers syndrome. Am J Hum Genet Suppl

1994;55(A92):519.

11. Neufeld EJ, Mandel H, Raz T, Szargel R, Yandava CN, Stagg A, Faure S, Barrett T, Buist N,

Cohen N. Localization of the gene for thiamineresponsive megaloblastic anemia syndrome, on

the long arm of chromosome 1, by homozygosity mapping. Am J Hum Genet 1997;61(6):1335-

1341.

12. Fleming JC, Tartaglini E, Steinkamp MP, Schorderet DF, Cohen N, Neufeld EJ. The gene

mutated in thiamineresponsive anaemia with diabetes and deafness (TRMA) encodes a

functional thiamine transporter. Nat Genet 1999;22(3):305-308.

13. Diaz GA, Banikazemi M, Oishi K, Desnick RJ, Gelb BD. Mutations in a new gene encoding a

thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome. Nat Genet

1999;22(3):309-312.

56


14. Labay V, Raz T, Baron D, Mandel H, Williams H, Barrett T, Szargel R, McDonald L, Shalata

A, Nosaka K, Gregory S, Cohen N. Mutations in SLC19A2 cause thiamine-responsive

megaloblastic anaemia associated with diabetes mellitus and deafness. Nat Genet 1999;22(3):

300-304.

15. Gass D. Desferrioxamine maculopathy. In: Klein EA, editor. Stereoscopic Atlas of Macular

Diseases. St. Louis, MO: The C.V. Mosby Company, 1987; 586-587.

16. Poggi V, Rindi G, Patrini C, De Vizia B, Longo G, Andria G. Studies on thiamine metabolism

in thiamineresponsive megaloblastic anaemia. Eur J Pediatr 1989;148(4):307-311.

17. Borgna-Pignatti C, Marradi P, Pinelli L, Monetti N, Patrini C. Thiamineresponsive anemia in

DIDMOAD syndrome. J Pediatr 1989;114(3):405410.

18 Schwingshandl J, Borkenstien M. Treatment of DIDMOAD syndrome with thiamine.

J Pediatr 1989;115:834.

19. Cagianut B, Hachstrasser P, Rhyner K. Diabetes juveniles, tapeto-retinal degeneration,

neurogene Schwerhörigkeit (Alström-syndrom), assoziiert mit einer kongenitalen

dyserythropoietischen Anämie typ III. Schweiz Med Wochenschr 1997;107: 446-450.

20. Valerio G, Franzese A, Poggi V, Tenore A. Long-term follow-up of diabetes in two patients with

thiamine-responsive megaloblastic anemia syndrome. Diabetes Care 1998;21(1):38-41.

21. Vora A, Lilleym J. Wolfram syndrome: mitochondrial disorder. Lancet 1993;342:1059.

22. Raz T, Barrett T, Szargel R, Mandel H, Neufeld EJ, Nosaka K, Viana MB, Cohen N. Refined

mapping of the gene for thiamine-responsive megaloblastic anemia syndrome and evidence for

genetic homogeneity. Hum Genet 1998;103(4):455-461.

23. Grill J, Leblanc T, Baruchel A, Daniel MT, Dresch C, Schaison G. Thiamine responsive anemia:

report of a new case associated with a thiamine pyrophosphokinase deficiency. Nouv Rev Fr

Hematol 1991;33(6):543-544.

24. Morimoto A, Kizaki Z, Konishi K, Sato N, Kataoka T, Hayashi R, Inoue F. A case of thiamine-

responsive megaloblastic anemia syndrome with nystagmus, cerebral infarction and retinal

degeneration. J Jpn Pediatr Soc 1992;96:2137-2145. References

57



Chapter 4

Ocular features in Rubinstein-Taybi syndrome: investigation of 24 patients andreview of the literature

Van Genderen MM, Kinds GF, Riemslag FCC, Hennekam RCM

Br J Ophthalmol 2000;84:1177-84

Institute for the Visually Impaired “Bartiméus”, Zeist, The Netherlands

MM van Genderen

GF Kinds

FCC Riemslag

Department of Pediatrics and Institute for Human Genetics,

Academic Medical Centre, Amsterdam, The Netherlands

RCM Hennekam



Ocular features in Rubinstein-Taybi syndrome:

investigation of 24 patients and review of the literature.

Abstract

Aims: To delineate the nature and frequency of ocular pathology inRubinstein Taybi syndrome (RTs).

Methods: Literature was searched for reports describing ocular symptoms inpatients with RTs. 24 RTs patients (out of a total of 73 Dutch knownRTs individuals) were selected for ophthalmological andelectrophysiological examination, selection being based only on thedistance between a patient’s residence and the place of investigation.

Results: Most frequently reported eye anomalies in the literature werelacrimal duct obstruction, corneal abnormalities, congenital glau-coma, congenital cataract, and colobomata. Abnormalities of almostany eye segment have been published in case reports. Ophthal-mological examination of 24 Dutch RTs patients showed a visualacuity <0.3 in five patients. The most frequently found eyeanomalies were nasolacrimal duct problems (six patients), cataract(six patients, four congenital), and retinal abnormalities (18patients). VEPs showed an abnormal waveform in 15 patients. Itwas possible to perform an ERG in 18 patients, of whom 14 wereabnormal (eight showed cone dysfunction, six cone-roddysfunction).

Conclusions: Ocular abnormalities occur in the majority of RTs patients and canbe remarkably diverse. The high frequency of retinal dysfunction(78%) has not been described before. With age, retinal as well aselectrophysiological abnormalities occur more frequently. In fourpatients no signs of retinal dysfunction were observed, indicatingphenotypic heterogeneity. Further cytogenetic and molecular exa-mination of the patients is needed before it becomes clear if this alsorepresents genetic heterogeneity. Because of the high frequency ofocular abnormalities, visual function tests and electrophysiologicalinvestigations should be performed in every RTs patient at regularintervals.

61


Rubinstein-Taybi syndrome (RTs) is a well known mental retardation-multiplecongenital anomalies syndrome, first described in 1957, but well delineated byRubinstein and Taybi in 1963.1 The incidence has been estimated to be one inevery 100 000 newborns.2 Reports of more than 1000 patients have been publishedworldwide.3 The syndrome is at least in part caused by microdeletions at chromo-some 16p13.3 or by mutations in the gene for the CREB binding protein (CBP),which is located at 16p13.3.4, 5 As anomalies disturbing the amount of functionalCBP are found in only 19% of patients,6 it remains possible that still other geneswill also prove to be causative. The major features of RTs are mental retardation,diminished growth, broad and sometimes medially deviated thumbs and big toes,and characteristic craniofacial abnormalities, consisting of microcephaly, down-ward slanted palpebral fissures, hypertelorism, long eyelashes, posteriorly rotatedears, beaked nose with the columella protruding well below the alae nasi, andpouting lower lip (see Fig 1). Data on ophthalmological findings are scarce. Thenumber of patients reported in ophthalmological journals is small, and only a fewwere published in the last decade.7–11 The only exception is the paper by Brei andco-workers,8 which provide a review of glaucoma and related conditions in RTs.However, all other ophthalmological features were not described, and no series ofpatients were personally investigated. This prompted us to perform a detailedophthalmological examination of a group of well defined Dutch patients with RTs,and to compare the results with those from literature.

Materials and methods

Literature reviewThe international literature was searched as completely as possible for reportsdescribing ocular symptoms in patients with RTs. For reports before 1990, abibliography was used;12 for the more recent literature, a Medline search wasperformed. Most papers were dealing with symptoms in a single patient only, andmost reviews paid only limited attention to ophthalmological features. Onlyreports describing patients in whom the diagnosis RTs was beyond doubt wereincluded. In total, 373 publications describing features in one or more patientswith RTs were available.

PatientsIn the Netherlands 73 patients with RTs are known (Hennekam RCM,unpublished data, 1999). Major non-ocular symptoms in 45 of the patients havebeen reported before2 and 13 others were tabulated elsewhere.13 These data were

62


63

Figure 1. (A) Characteristic facial Rubinstein-Taybi syndrome in a 13 year old female patient. (B) Downwardslanted palpebral fissures, long eyelashes, beaked nose, pouting lower lip, and mildly dysplastic and posteriorlyrotated ears. (C) Typical hand characteristics in the same patient with Rubinstein-Taybi syndrome. (D) Typicalfoot characteristics in the same patient.


in general in accordance with the data in other larger series of patients.14, 15 Twentyfour of the published patients were selected for incorporation in the study. Theselection was based solely on the distance between their homes and the institutewhere the investigation took place. All parents or guardians approved inclusion.The study was approved by the medical ethics committee of the Academic MedicalCentre in Amsterdam.

Ophthalmological investigationsComplete ophthalmic examinations were performed on all patients. Visual acuitywas assessed with two of the following methods: forced preferential looking,16

matching optotypes (HOTV),17 and Snellen visual acuity chart. Four patientscould be tested with preferential looking only, because of age and mental develop-ment. Visual fields were tested with confrontation techniques. Only a limitednumber of patients were able to match and name colours. Therefore, testing withcolour plates was not attempted. Pupillary responses, eye motility, strabismus, andthe external aspects of the eyes and adnexa were assessed. Slit lamp examination was performed in all patients. Funduscopy and retinoscopywere performed after cycloplegia except in one patient, in whom installation of eyedrops was refused. In this patient examination of the optic disc was possible, but areliable assessment of the macular region could not be made. Applanationtonometry was performed in four patients only; in most patients we did notattempt tonometry because of the risk of losing cooperation.

Electrophysiological investigationsVisual evoked potentials (VEPs) were elicited with pattern onset stimuli,18 andrecorded in all patients. Check sizes of 60', 30', 15', and 5' were used to evokepattern onset responses. Electroretinography (ERG) was measured withoutsedation. Cooperation was insufficient in six patients for this examination, leaving18 patients investigated. ERGs were recorded with Henkes contact lens electrodesor, in less cooperative patients, DTL electrodes. In these mentally handicappedsubjects measurements according to the standard ISCEV protocol were notpossible, since the ISCEV protocol is lengthy and requires the patients to be in thedark for a long period of time. Therefore, we reduced the ERG protocol to theabsolute minimum: a standard ISCEV flash photopic response (with a bluebackground of 10 cd/m 2) and a scotopic response (-2.0 ND (Kodak Wratten filter)standard flash in the dark after 10 second dark adaptation). The stimulatorconsisted of an adapted dome, larger than normal, with mirrors, which allowed for

64


a hospital bed with the subject lying down to be put below it. In this way, good(eye) contact of the patient with his or her parents remained possible. Therecording equipment was kept in a separate room. For this type of stimulation thenormative values of the amplitudes are: scotopic (b-peak to onset) 66 (SD 27) µV;photopic (b-peak to a-peak) 137 (64) µV; mixed response (to the standard flash inthe dark) 347 (94) µV (FCC Riemslag, unpublished data). Recording of the mixedresponse was possible in only nine patients.

Results

Literature reviewEighty one reports describing 207 patients in suffcient detail were searched forophthalmological features; 117 patients were found to have ocular abnormalities(Table 1). External, mainly (peri)ocular findings adding to the characteristic facialappearance were described in the majority of published cases: downward slanted

65


Symptom Number with References symptom

Lacrimal duct problems 21 15, 19–25 High myopia 11 1, 7, 21, 26–32 Nystagmus 5 22, 28, 33–35 Pupillary abnormalities 3 22, 36, 37 Microcornea/microphthalmia 6 22, 38–42 Corneal abnormalities 23 1, 21, 30, 31, 40, 43–49 Congenital glaucoma 31 15, 25, 26, 41, 51, 52, 54–67 Iris atrophy 2 30, 68 Congenital cataract 1 5 15, 21, 24, 26, 27, 33, 38, 39, 41, 53, 69, 70 Ectopia lentis 1 37 Microphakia 1 28 Coloboma (iris, lens, retinal, and/or optic nerve) 39 1, 15, 19, 24, 27, 28, 39, 42, 48, 51, 72–83 Chorioretinal dystrophy 6 32, 84–88 Optic atrophy 7 1, 20, 35, 69, 89, 90 Optic disc abnormalities 4 34, 63, 91, 92

Table 1. Ocular symptoms in patients with Rubinstein-Taybi syndrome; 117 out of 207 patients (81 case reports)

66


lacr.

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OD

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67


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palpebral fissures, hypertelorism, epicanthus, long eyelashes, ptosis (eitherunilateral or bilateral), and strabismus (Fig 1). The mentioning of these featuresdepended heavily on the thoroughness of the descriptions of the patients by thedifferent authors. Therefore, these features are not specifically mentioned in thetable, and tabulation was restricted to severe problems in visual acuity, lacrimalapparatus anomalies, and intraocular findings.Lacrimal duct problems (21 cases), corneal abnormalities (that is, megalocorneawithout glaucoma, opacities, keratoglobus, sclerocornea) (23 cases), congenitalglaucoma (31 cases), congenital cataract (15 cases), and colobomata (39 cases) werethe most frequently described serious ocular abnormalities. No incidence figuresof the various abnormalities can be extracted from these case reports, owing toascertainment bias and publishing of incomplete data of ophthalmologicalinvestigations in the majority of cases. In 1990, Rubinstein 14 reviewed 571individuals with RTs gathered both from literature and from personal investiga-tions or personal communications with other physicians worldwide. Refractiveerrors were seen in 56% of cases, strabismus in 71%, ptosis in 29%, and lacrimalduct anomalies in 37% of cases. No data regarding the frequency of other eyeanomalies were provided.

Findings in 24 Dutch patientsAll major results are summarised in Tables 2 and 3. Five patients (20%) werevisually handicapped with a visual acuity equal to or below 0.3. Visual fields werenormal in all patients. Strabismus was found in 17 patients (67%). Refractiveerrors were common: six patients (25%) were highly myopic (excess of -6dioptres). Photophobia occurred in 11 patients: in four, possible causes were found(cataract in two, corneal herpes infection in one, pterygium in one case), but inseven the photophobia could not be explained by anterior segment abnormalities.Nasolacrimal duct obstruction occurred in six cases (25%), in two of them unila-terally. Three cases had severe congenital ocular abnormalities: congenital cataractand colobomata, congenital glaucoma, and unilateral microphthalmia withcongenital cataract, respectively. Two other cases had congenital cataracts that didnot cause significant visual disability and were therefore left untreated. Twopatients had presenile or secondary cataracts. On funduscopy, macular abnormal-ities were found in 18 patients (75%). Thirteen cases had mild macularabnormalities, that is, abnormal or absent reflexes, increased reddening of thefoveal area, or unusual distribution of pigmentation in the posterior pole (Fig 2).Five cases had more severe macular abnormalities. Pigmentary macular changes

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and atrophy were seen in patients “n”, “t” (Fig 3), and “u”. Two of these patientswere myopic, but the degree of macular changes was much more severe thanexpected with regard to age and rate of myopia. Patient “r” showed lacquer cracksas a result of high myopia in her left eye, but her right eye, which had only mildmyopia, also showed pigmentary changes. Patient “x”, who had a myopia of -22dioptres, had severe myopic degeneration. Patient “v” had a small chorioretinal

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Figure 2. Seven year old male patient with Rubin-stein - Taybi syndrome, showing mild macular abnor-malities: unusual distribution of pigment, red fovealarea.

Figure 3. Fundus of 34 year old patient with Rubin-stein-Taybi syndrome with retinal pigment epithelial changes.

Figure 4. Right fundus of 37 year old female patientwith Rubinstein-Taybi syndrome showing a pale and excavated optic disc and a small chorioretinalcoloboma.


coloboma (Fig 4). Visual evoked potentials showed an abnormal waveform in 15patients (62.5%) with a missing striate or foveal component. In Figure 5 theresponses of a subject with normal vision and fundus (VODS >1.0), a patient withRTs, and a patient with achromatopsia are shown for comparison. As is knownfrom previous research,93–95 the first (normal) subject shows a response composedof a striate component (predominantly negative, peak latency at approximately 110ms), and a extrastriate component (positive negative, the positivity at about 80 ms,the negativity at 160–200 ms). As the check sizes become smaller, the striatecomponent becomes more prominent, the extrastriate component diminishes inamplitude. In the (characteristic) response of an RTs patient, only an extrastriate

70

Figure 5. Pattern onset visual evoked potentials of a normal subject (left), patient with RTs (middle), andpatient with achromatopsia (right).

*More than 1 dioptre; †nm = not mentioned

Table 4. Frequency of external ocular abnormalities in our patients compared with 571 patients reviewed by

Rubinstein14 and 50 patients from a questionnaire study of Stevens15

Nasolacrimal Refractive duct

Strabismus error* obstruction Cataract† Coloboma† Glaucoma†

Rubinstein (n=571) 71% 56% 37% nm nm nm Stevens (n=50) 48% 38% 30% 6% 6% 2% Our patients (n=24) 71% 67% 25% 20% 8% 4%

component is seen (positive negative, P100, N180); this response diminishes inamplitude with decreasing check size. For the achromat, only the biggest checksizes (60') evoke a significant response, also only containing an extrastriatecontribution (note the differences in the voltage scale). Of the 18 ERGs recorded14 (78%) showed anomalies. Eight ERGs showed decreased cone responses andsix ERGs showed decreased cone and rod responses.

Discussion

The great variety of ocular abnormalities, involving all eye segments, that occursin Rubinstein-Taybi syndrome is remarkable, and provides another indication forthe ubiquitous functions of CBP. Two previous studies of RTs patients14, 15 showeda frequency of ocular abnormalities comparable to the frequency of abnormalitiesin our patients (Table 4). However, they did not report on retinal dysfunction, andin literature only six cases with possible retinal dystrophy were published.32–88 Inthese cases fundus abnormalities such as pigmentary changes, absent fovealreflexes, and/or a dystrophic retina were described. In two patients an ERG wasmade32, 87; one was attenuated.32 In one patient88 a VEP was made which was statedto be normal. However, the authors did not mention whether or not the VEP wasa flash, pattern onset, or pattern reversal VEP. The abnormal waveforms we reporton here can only be seen in pattern onset VEPs, with the standard pattern reversalresponses being normal. Approximately a third of the patients from the literature were examined bypaediatricians. Routine examination by an ophthalmologist was mentioned only ina few cases. Most patients seemed to have been assessed by an ophthalmologistonly if they had obvious ocular abnormalities. This may account for the higher fre-quency in literature of external abnormalities (that is, cornea, congenital glauco-ma) compared with fundus changes. Furthermore, as in our patients, funduschanges may have been mostly mild, not leading to a diagnosis of retinal dystrophywithout ERG measurements. According to the World Health Organization definition, a person is consideredvisually handicapped if the person’s best corrected binocular visual acuity is at orbelow 0.3. In this study, five patients were visually handicapped (21%). In addition,three other patients had a low visual acuity in only one eye. Although congenitalor presenile cataract occurred frequently in our patients (25%), only in one patientthe visual handicap was caused by cataract. In contrast with the low incidence in literature, 78% of the present patientsshowed signs of retinal dysfunction as measured by ERG. In general, an abnormal

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Fundus abnormalities ERG VEPAge none mild severe normal cone cone rod normal abnormal

<15 4/13 (31%) 9/13 (69%) 0 3/9 (33%) 4/9 (44%) 2/9 (22%) 6/13 (46%) 7/13 (54%) 15–30 1/5 (20%) 2/5 (40%) 2/5 (40%) 1/5 (20%) 1/5 (20%) 3/5 (60%) 2/6 (33%) 4/6 (67%) >30 0 2/5 (40%) 3/5 (60%) 0/4 (0%) 3/4 (75%) 1/4 (25%) 1/5 (20%) 4/5 (80%)

Table 5. Increasing incidence of fundus, ERG, and VEP abnormalities with age

*<1 SD below the mean. **<2 SD below the mean.

Table 6. ERG values of 24 patients with Rubinstein-Taybi syndrome

ERGPatient Age Photopic Scotopic Mixed

Normal values (SD) 137 (64) (n=18) 66 (27) (n=18) 347 (94) (n=18) a 4 nm nm nm b 6 7* 0** nm c 6 nm nm nm d 7 69* 53 nm e 7 20* 33* nm f 8 45* 77 218* g 9 nm nm nm h 10 80 58 305 i 12 82 110 266 j 14 55* 94 132* k 15 35* 56 nm l 15 86 82 222* m 15 nm nm nm n 19 39* 0** nm o 21 nm nm nm p 24 45* 34* 112** q 24 53* 0** 95** r 29 53* 99 137** s 30 75 51 280 t 34 27* 138 222* u 36 30* nm nm v 37 36* 0** nm w 43 39* 52 nm x 44 nm nm nm

ERG is caused by abnormally functioning of photoreceptors, bipolar cells, andMüller cells. Abnormally functioning of the retinal pigment epithelium (RPE) canalso result in ERG disturbances, as the RPE is necessary for normal functioningof the photoreceptors. All abnormal ERGs showed a decreased cone response,indicating cone dysfunction. Seven patients had unexplained photophobia.Photophobia is often seen in patients with cone dysfunction. The abnormalwaveform in VEP testing was assumed to be caused by the abnormally functioningretina. A normal pattern onset VEP has a striate component that for the largestpart is determined by foveal function. This component was missing in allabnormal VEPs. If there are no normally functioning cones, there will be anabnormal or absent striate component in the VEP. A similar waveform can befound in the VEPs of patients with total absence of functioning cones- that is,achromatopsia.96, 97

As demonstrated in Figure 5 in which the responses in individuals with RTssubjects are compared with the responses of achromat individuals, the responsesshare the absence of a striate component. The extrastriate component in the RTsindividuals is evoked for various check sizes, whereas in the achromat people it isclassically found only for the largest check sizes. This suggests in general a bettervisual acuity for the RTs patients compared with the achromats, which iscorroborated by the presently reported findings. In four patients the VEP andERG were not in accordance: three times the ERG was subnormal with a normalVEP, and once a normal ERG was registered with an abnormal VEP. Because anERG measures the overall activity of the retina, locally abnormally functioningcones can give rise to an abnormal VEP without significant influence on the ERG.On the other hand, if the dysfunction of the photoreceptors is widespread butmacular vision is preserved, the ERG can be grossly abnormal with a normalVEP.100, 101 In the majority of patients in which an ERG could be measured, theERG and VEP were in accordance (78%). Furthermore, the abnormal VEPs ofpatients with normal ERGs showed the same extrastriate pattern as the VEPs ofpatients with attenuated ERGs. Therefore, in patients in which an ERG could notbe performed, we considered the abnormal VEP a sign of retinal dysfunction. Theincidence of abnormal ERGs and VEPs as well as of retinal abnormalitiesincreases with age (Table 5), suggesting a progressive retinal dystrophy. However,only future careful follow up will allow any firm conclusions regarding progressionwith age. Retinal dystrophy comprises a genetically heterogeneous group ofdisorders, which can occur in a wide variety of systemic disorders.100, 101 The

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occurrence of retinal dystrophy in RTs means that expression of the defective genecausing RTs is also present in the retina. In 18 patients, funduscopy,electrophysiology, or both, showed signs of retinal dysfunction, but in fourpatients both were normal. ERG changes occur very early in retinal dystrophy,most often before any retinal changes are seen (Table 6). Hence, it is less likely thatin the latter four patients retinal dystrophy will yet develop, but three of thesepatients are still children and without follow up this can not be concluded withcertainty. If retinal dystrophy will indeed not arise, this indicates phenotypicheterogeneity with regard to retinal function. It is uncertain whether this canmerely be explained by the pleiotropic expression of the abnormal CBP gene inRTs, or is an indication for genetic heterogeneity in RTs. In a limited number ofthe present patients cytogenetic studies using fluorescence in situ hybridisationtechniques, and molecular studies have been performed. All (seven) cases in whomeither a submicroscopic deletion or a mutation in the CBP gene have been founduntil now, also showed the electrophysiological abnormalities (data not shown).This suggests that the presence or absence of the electrophysiologicalabnormalities may be indicative for the presence or absence of abnormalitiesinvolving the CBP gene, and suggests genetic heterogeneity. However, beforesuch conclusion can be drawn all patients, both with and withoutelectrophysiological abnormalities, should be investigated cytogenetically usingFISH for microdeletions at chromosome 16p13.3 and molecularly for mutationsin the CBP gene. Such studies are in progress.

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Chapter 5

The EEM syndrome, a ten year follow-up of two new patients

van Genderen MM1, Dixon MJ 2, Riemslag FCC 1,Stilma JS 3, Meire FM4

Submitted

1 Bartiméus Institute for the Visually Impaired, Zeist, The Netherlands

2 Faculty of Life Sciences, University of Manchester, United Kingdom3 Department of Ophthalmology, University Medical Center Utrecht, The Netherlands 4 University Children’s Hospital Koningin Fabiola, Brussel en University Hospital Gent, Belgium



The EEM syndrome, a ten year follow-up of two

new patients

Abstract

The EEM syndrome (Ectrodactyly, Ectodermal Dysplasia, and MacularDystrophy) is a rare inherited disorder affecting the limbs, eyes, hair and teeth.Affected patients have hypotrichosis, malformations of the distal extremities suchas syndactyly and ectrodactyly, and retinal abnormalities mainly of the posteriorpole. In addition, there are narrow teeth, missing teeth, or both. Recently it wasascertained that EEM is caused by homozygous mutations in CDH3, encoding P-cadherin.We describe a brother and sister with the EEM syndrome, born from unaffectedparents. Of their four healthy siblings, one sister had teeth abnormalities, whichmay be a mild manifestation of the syndrome in a carrier. The patients already had extensive retinal abnormalities in infancy. The retinalappearance was consistent with a congenital abnormality of Bruch’s membrane,rather than a macular dystrophy. Follow-up of more than ten years showed mini-mal changes in fundoscopic appearance and visual functions. The ERG in bothchildren remained normal, indicating normal photoreceptor function.Ophthalmologically, the EEM syndrome appears distinct from hypotrichosis withmacular dystrophy (HJMD), a syndrome also caused by mutations in CDH3.

Key words

EEM syndrome, retinal dysplasia, ERG, Bruch’s membrane, HJMD, CDH3

Introduction

In 1956, Albrectsen and Svendsen described a previously unknown syndrome intwo Danish children, a brother and sister.1 The children had syndactyly, sparsehair, and retinal abnormalities interpreted as a retinal degeneration. Their psycho-motor development was normal. Ohdo2 first used the acronym EEM in 1983, in adescription of an inbred family with the syndrome. A total of twelve patients have been described so far, with significant clinicalvariability of the symptoms1-5 (Table 1). All patients had retinal abnormalities con-sisting of pigmentary alterations or atrophic lesions of the posterior pole, somealso had peripheral lesions. (Table 2). The visual functions of some patients

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deteriorated over several years,4,5 while in others they seemed to be fairly stable.1,2,3

Several patients were born from consanguineous marriages,1,2,4 which suggestedautosomal recessive inheritance. A recent study by Kjaer6 of two previously reported families showed homozygousmutations in CDH3, encoding P-cadherin, to be the cause of EEM. P-cadherin isan important component in maintaining cell-cell adhesions at adherens junctionsthrough Ca2+ -dependent homophilic interactions.7 It is thought to be involved inthe regulation of both hair8 and retinal9 development.

In this report we describe two children, a brother and a sister, who presented withthe EEM syndrome. We first examined them as infants, and present follow-updata of more than 10 years on their visual functions, tested both psychophysicallyand electrophysiologically.

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author sex hands feet hypotrichosis teeth

Albrectsen f syndactyly normal + small, widely spacedm ectrodactyly webbing + normal

Ohdo f syn/ ectrodactyly poly/syndactyly + nmf ectrodactyly syndactyly + small, widely spacedf syn/ ectrodactyly normal + nmm ectrodactyly syn/ ectrodactylyv + nmm ectrodactyly syn/ ectrodactyly + nm

Hayakawa f ectrodactyly nm + small, irregular, oligodontiaBalarin Silva m syndactyly nm + small, widely spaced

m webbing nm +Senecky f syndactyly nm + normal

m syndactyly nm + normalthis study f syn/ ectrodactyly normal + small, widely spaced, oligodontia

m syn/ ectrodactyly normal + small, widely spaced

f, female; m, male; nm not mentioned

Table 1. EEM syndrome, summary of clinical features (except ocular) of the 6 described families

Case report

Clinical findingsIn 1990, we saw a one year old girl, the fifth child of unrelated parents. She wasborn at term after an uneventful pregnancy, with a birth weight of 4180 gm. Shehad extensive abnormalities of both hands (see Figure 1). Her scalp hair was sparseand she had scanty, very blond eyelashes and eyebrows (Figure 2). When she grewolder, her dentist observed three missing teeth; furthermore her teeth were rathernarrow and widely spaced (Figure 3). Her mental development was normal. Thefindings in the girl were suggestive of the EEM syndrome.In 1992, her brother was born, also with features of the EEM syndrome withextensive hand malformations (see Figure 4). He also had very sparse scalp hairand very blond eyelashes and eyebrows. His teeth were widely spaced but normalin number. The girl had three older brothers and one older sister without any congenitalabnormalities, except for nine missing teeth in the sister.

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VA1, visual acuity at first examination; VA2, visual acuity at follow up examination; M, of the macula; P, of the(mid)periphery; nm, not mentioned

Table 2. ocular features in the EEM syndrome

author VA1 VA2 fundus ElectrophysiologyOD OS (age) OD OS (age) RPE alterations atrophic lesions ERG EOG

Albrectsen 0.16 0.1 (6) M,P 0.12 (4) M,P

Ohdo nm (16) M Mnm (10) Mnm (39) M Mnm (45) M Mnm (42) M M

Hayakawa 0.07 0.01 (30) 0.03 0.01 (41) M M subnormal flatBalarin Silva 0.25 0.25 (23) 0.1 0.1 (33) M normal normal

0.3 0.3 (25) 0.3 0.1 (35) M normal normalSenecky nm (12) M M abnormal

nm (9) M M abnormalthis study 0.1 0.1 (1) 0.05 0.05 (4) M,P normal

0.08 0.08 (0.7) 0.05 0.05 (12) M,P normal normal

Ocular findingsOphthalmologic examination in the girl showed a visual acuity of 0.1, tested at ageone, two, and five with a test appropriate for age; her visual acuity was 0.06 at nineand fourteen years. She had a divergent squint and nystagmus. Color vision testingat age seven and thirteen was normal, as were her peripheral visual fields and darkadapted thresholds. Biomicroscopy was normal. Fundoscopy revealed normaldiscs and blood vessels. The retina showed severe pigmentary alterations andatrophic lesions in the macular area (Figure 5). Retinal pigment epithelium (RPE)disturbances were also observed in the midperiphery with hyperpigmentations,white deposits, and the aspect of peau d’orange (Figure 6). Subretinal fibrosis wasespecially noticed around the optic discs). Flash electroretinography (ERG)recordings according to ISCEV standards10 were made at age two, and repeated atage 14. The scotopic, mixed, and photopic responses of both recordings werewithin normal limits.The boy had a visual acuity of 0.08 at ten months of age, 0.1 at four and sevenyears, and 0.05 at ten and twelve years. He had a divergent squint and nystagmus.Color vision testing at seven and twelve years was normal, as were his peripheralvisual fields and dark adapted thresholds. Biomicroscopy was normal; fundoscopyshowed widespread pigmentary changes with the most obvious changes in themacular area. Discs and vessels were normal (Figure 7). ERG testing at age 12 showed normal scotopic, mixed and photopic responses.The electro-oculogram (EOG11) showed a normal Arden ratio of 2.0 in the righteye, and 2.4 in the left eye.

Discussion

Including the two patients in this study, only fourteen patients, have beendescribed with the EEM syndrome since 1956.1-5 All patients had distal limbmalformations, hypotrichosis, and macular pigmentary changes; their visualacuities varied from 0.05 to 0.3. The reports showed significant clinical variabilityin the EEM phenotype (Table 1). Several patients had major hand malformationswith missing phalanges or even whole fingers; some also had foot abnormalities.In contrast, others had relatively minor hand abnormalities and normal feet. Ninepatients had teeth abnormalities. No relationship appears to exist between theextent of the hand malformations and the ocular features

The two siblings in this study are the first patients to have been examined ininfancy; at that time retinal abnormalities were already extensive. Follow-up of

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Figure 1. Hand abnormalities of the girl at age one. The right hand showed missing distal phalanges of the second, third,and fourth fingers, a doubling of the proximal phalanx of the second, and syndactyly of the second and third fin-ger; the left hand had syndactyly of the first, second, third, and fourth finger and a doubling of the proximal phalanx of the second

Figure 2.Girl at age 7, showing hypotrichosis

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Figure 3. Dental abnormalities of the girl. There are two missing upper incisors (1II and 2II), one missing lower incisor(4II). Furthermore the teeth are rather narrow and widely spaced.

Figure 4. Hands of the boy at nine months of age. The malformations of his right hand consisted of syndactyly of thethird and fourth fingers with missing distal phalanges. His left hand had syndactyly of the second and third fingers with a missing distal phalanx of the fourth finger.

more than 10 years showed only minimal changes in their retinal appearance. Thepatients had macular as well as peripheral RPE disturbances; the latter were alsodescribed in the original Danish family.1 The early presence of retinal abnor-malites indicates a congenital dysplasia rather than a retinal dystrophy. Thecongenital abnormality could well be associated with Bruch’s membrane pathologybecause of the similarities with the fundus appearance in severe pseudoxanthomaelasticum.12-14 The ERG in both children was normal, indicating normal photo-receptor function, which also argues against a retinal dystrophy.

Recently, it was ascertained that distinct CDH3 mutations cause EEM.6 CDH3mutations also cause hypotrichosis with juvenile macular dystrophy (HJMD),15-23 acondition closely resembling EEM, but without teeth or hand abnormalities.CDH3 encodes P-cadherin, one of the three classical (E-, N-, and P-) cadherins.Cadherins are a family of glycoproteins involved in Ca++-dependent cell-celladhesion. Classical cadherins play a major role in embryonic development, tissueformation, and cell growth and differentiation.24 In a mouse model, CDH3 isexpressed during embryonal development in craniofacial structures, and the foreand hind limbs.6

Since the initial identification of CDH3 mutation as the cause of HJMD, a total

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Figure 6. Posterior pole of the boy with widespread pigmentarychanges, white deposits, and normal discs and vessels.

Figure 5. Posterior pole of the girl, showing severe pigmentaryalterations and atrophic lesions in the macular area,and subretinal fibrosis around the optic disc.

of seven mutations in 11 separate families have been reported.18-21 All mutationsseem to impair markedly the function of P-cadherin or result in the absence of functional P-cadherin expression. However, considerable phenotypic hetero-geneity and lack of phenotype-genotype correlation was found, even within singlefamilies carrying the same mutation, suggesting other, possibly genetic, modifiertraits.20

HJMD and EEM may represent phenotypic heterogeneity of the samesyndrome.6, 22 However, the ophthalmological abnormalities described in HJMDseem different from those in our patients. The retina in HJMD initially may looknormal,16 or have some asymptomatic pigmentary depositions confined to themacula,21 but subsequently a progressive macular degeneration develops, some-times even leading to legal blindness.15, 16, 18 Furthermore, the ERG in HJMD isoften already abnormal at a young age, indicating photoreceptor dysfunction.18-21

In contrast, the ERGs of our patients remained normal over a period of ten years. Follow-up on the ophthalmological features of EEM is described in three otherstudies;3-5 in one of those, the retinal appearance also hardly changed over a periodof ten years.4 On the other hand, the patients described by Balarin Silva andSenecky showed progression of their retinal lesions and deterioration ofelectrophysiological function. Remarkably, these patients had the least severe handabnormalities of the EEM families described, consisting only of syndactyly (seeTable 1). One could argue that these patients have a HJMD rather than an EEMphenotype, because of the absence of ectrodactyly, combined with progressivemacular degeneration with initially good vision. Support for this suggestion alsocomes from the fact that the mutation found in the Brazilian EEM kindred(829delG)6 is the same as previously found in a Turkish HJMD family.20 On theother hand, the Brazilian brothers did have small and widely spaced teeth. Noother HJMD patients have been described with teeth malformations, although theoften subtle dental abnormalities as seen in EEM may be overlooked withoutspecialist dental examination. The intermediate phenotype in this kindred maytherefore be an indication that HJMD and EEM are in fact one syndrome withphenotypic heterogeneity.

A function of P-cadherin in Bruch’s membrane development has not beendescribed, but P-cadherin mRNA is highly expressed in the retinal pigmentepithelium (RPE), 23 from which Bruch’s membrane is partly derived.25 In mice,loss of P-cadherin does not result in obvious hair or ophthalmologicalabnormalities. It has therefore been suggested that E-cadherin expression may be

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turned on in P-cadherin null mice, or that RPE cells may express other cadherinswhich are capable of compensating for the loss of P-cadherin.23

E-cadherin may also play a role in determining the phenotypic differencesbetween HJMD and EEM.6 CDH1, encoding E-cadherin, is known to haveredundant functions. Compensation of CDH1 for the CDH3 loss of function mayultimately determine if a patient will have HJMD or EEM. Expression differencesand redundant functions of other cadherins may not only explain the differentphenotypes of HJMD and EEM, but also the phenotypic differences betweenhumans and mice carrying mutations in orthologous genes.18

In the study of Balarin Silva,4 a brother and a cousin of the patients had isolatedhand abnormalities. Kjaer6 describes mild bilateral syndactyly and widely spacedteeth in one heterozygous individual. In our study, one sister had teethabnormalities. These abnormalities in relatives may represent mild manifestationsof the syndrome in heterozygotes.

The siblings in this study are the thirteenth and fourteenth patient with EEM tobe described in 49 years, and the first ones in which the diagnosis was establishedin infancy. Molecular analysis of this family is currently under way.

Acknowledgements

We like to thank dr. J. van Twillert for his description of the teeth abnormalitiesand for the dental X-ray.

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References

1. Albrectsen B, Svendsen IB. Hypotrichosis, syndactyly and retinal degeneration in two siblings.

Acta Derm Venereol 1956;2:96-101

2. Ohdo S, Hirayama K, Terawaki T. Association of ectodermal dysplasia, ectrodactyly, and

macular dystrophy: the EEM syndrome. J Med Genet 1983;20:52-57.

3. Hayakawa M, Kato K, Nakajima A, Yamauchi H. Association of ectodermal dysplasia,

ectrodactyly and macular dystrophy: EEM syndrome (case report). Ophthal Paediatr Genet

1989;10:287-292

4. Balarin Silva V, Simões AM, Marques-de-Faria AP. EEM syndrome report of a family and results

of a ten-year follow-up. Ophthalmic Genet 1999;20:95-99

5. Senecky Y, Halpern GJ, Attias J, Shohat M. Ectodermal dysplasia, ectrodactyly and macular

dystrophy (EEM syndrome) in siblings. Am J Med Genet 2001;101:195-197.

6. Kjaer KW, Hansen L, Schwabe GC, Marques-de Faria AP, Eiberg H, Mundlos S, Tommerup N,

Rosenberg T. Distinct CDH3 mutations cause ectodermal dysplasia, ectrodactyly, macular

dystrophy (EEM syndrome). J Med Genet 2005;42:292-8.

7. Yagi Y, Takeichi M: Cadherin superfamily genes: functions, genomic organization and

neurological diversity. Genes Dev 2000; 14:1169-1180.

8. Muller-Rover S, Tokura Y, Welker P, Furukawa F, Wakita H, Takigawa M, Paus R. E- and P-

cadherin expression during murine hair follicle morphogenesis and cycling. Exp Dermatol.

1999;8:237-246.

9. Riehl R, Johnson K, Bradley R, Grunwald GB, Cornel E, Lilienbaum A, Holt CE. Cadherin

function is required for axon outgrowth in retinal ganglion cells in vivo. Neuron.1996;17:837-

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Ophthalmol 1995;89:199-210.

11. Marmor MF, Zrenner E. Standard for clinical electro-oculography. Documenta Ophthalmol

1993;85:115-124.

12. Giuffre G. The pathogenesis of peau d’orange and salmon spots. Metab pediatr Syst Ophthalmol

1987;10:95-98.

13. Shiraki K, Moriwaki M, Matsumoto M, Khono T, Miki T. Hypofluorescent spots in indocyanine

green angiography of peau d’orange and fundus. Int Ophthalmol 1997;21:43-50.

14. Lafaut BA, Leys AM, Scassellati-Sforzolini B, Priem H, De Laey J.J. Comparison of fluorescein

and indocyanine green angiography in angioid streaks. Graefes Arch Clin Exp Ophthalmol

1998;236:346-353.

15. Marren P. Wilson C. Dawber RP, Walshe MM. Hereditary hypotrichosis (Marie-Unna type) and

juvenile macular degeneration (Stargardt’s maculopathy). Clin Exp Dermatol 1992;17:189-191.

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16. Souied E, Amalric P, Chauvet M-L, Chevalier C, Le Hoang P, Munnich A, Kaplan J. Unusual

association of juvenile macular dystrophy with congenital hypotrichosis: occurrence in two

siblings suggesting autosomal recessive inheritance. Ophthalmic Genet 1995;16:11-15.

17. Becker M, Rohrschneider K, Tilgen W, Weber BH, Volcker HE. Familial juvenile macular

dystrophy with congenital hypotrichosis capitis. Ophthalmologe 1998;95:233-240.

18. Sprecher E, Bergman R, Richard G, Lurie R, Shalev S, Petronius D, Shalata A, Anbinder Y,

Leibu R, Perlman I, Cohen N, Szargel R. Hypotrichosis with juvenile macular dystrophy is

caused by a mutation in CDH3, encoding P-cadherin. Nat Genet 2001;29:134-146.

19. Indelman M, Bergman R, Lurie R, Richard G, Miller B, Petronius D, Ciubutaro D, Leibu R,

Sprecher E. A missense mutation in CDH3, encoding P-cadherin, causes hypotrichosis with

juvenile macular dystrophy. J Invest Dermatol 2002;119:1210-1213.

20. Indelman M, Hamel CP, Bergman R, Nischal KK, Thompson D, Surget MO, Ramon M,

Ganthos H, Miller B, Richard G, Lurie R, Leibu R, Russel-Eggitt I, Sprecher E. Phenotypic

diversity and mutation spectrum in hypotrichosis with juvenile macular dystrophy. J Invest

Dermatol 2003; 121:1217-1220.

21. Indelman M, Leibu R, Jammal A, Bergman R, Sprecher E. Molecular basis of hypotrichosis with

juvenile macular dystrophy in two siblings. Br J Dermatol 2005;153:635-638.

22. Warburg M. Macular dystrophy and hypotrichosis: the EEM-Albrectsen syndrome. Ophthalmic

Genet 1995;16:177-178.

23. Xu L, Overbeek PA, Reneker LW. Systemic analysis of E-, N-, and P-cadherin expression in

mouse eye development. Exp Eye Res 2002;74:753-760.

24. Gumbiner BM. Cell adhesion: the molecular basis of tissue architecture and morphogenesis.

Cell 1996;84:345-57.

25. Takei Y, Ozanics V. Origin and development of Bruch's membrane in monkey fetuses: an

electron microscopic study. Invest Ophthalmol 1975;14:903-16

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Chapter 6

Misrouting and foveal hypoplasia without albinism

van Genderen MM,1 Riemslag FCC,1 Schuil J,1 Hoeben FP,1

Stilma JS,2 Meire FM 3

Submitted

1 Bartiméus Institute for the Visually Impaired, Zeist, The Netherlands

2 Department of Ophthalmology, University Medical Center Utrecht, The Netherlands3 Department of Ophthalmology, University Children’s Hospital Koningin Fabiola, Brussel

and Academic Hospital Gent, Belgium



Misrouting and foveal hypoplasia without albinism

Abstract

Background/aims : To present the clinical and electrophysiological characteristics of

three female patients with misrouting and foveal hypoplasia. Allpatients were darkly pigmented, without iris transillumination orfundus hypopigmentation. One of the patients had primary ciliarydyskinesia and situs inversus totalis (Kartagener syndrome).

Methods: The patients underwent ophthalmological examination, fundusphotography, and optical coherence tomography (OCT). Visualevoked potentials (VEP) were recorded and calculation of thechiasmal coefficient was performed to detect misrouting.

Results: The patients had a visual acuity of 0.1 to 0.2 and nystagmus. Fundusphotography and OCT showed foveal hypoplasia, with a vascularpattern different from that found in albinism. The monopolar anddifferential VEP recordings, as well as the chiasmal coefficient, werepositive for misrouting.

Conclusion: Based on the investigations in these patients we propose thehypothesis that foveal hypoplasia and misrouting exist as a distinctentity, and do not comprise the exclusive hallmark of albinism. Themisrouting in the patients may have been caused by defective geneswhich are involved in chiasm formation. Furthermore, the findingssuggest that misrouting may exert a retrograde influence on fovealdevelopment.

Key words: albinism, misrouting, foveal hypoplasia, Kartagener, optic chiasm

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Albinism is a heterogeneous genetically determined disorder of melanin synthesisin which either the eyes (ocular albinism), or the eyes, skin, and hair may beaffected (oculocutaneous albinism). Clinical signs show considerable variability inindividual patients (for review, see Russell-Eggitt1). The optic discs show an ab-normal course of retinal vessels consisting of an initial nasal deflection followed byan abrupt divergence and reversal of direction to form the temporal vasculararcades.2

In albinism, a far greater number of fibres from the temporal retina cross themidline and project contralaterally than in normals. This misrouting can bedetected by recording multichannel visually evoked potentials (VEPs).3 Thegeneral assumption is that if misrouting is detected, the diagnosis of albinism isproven unequivocally. Foveal hypoplasia in albino patients shows a strong correla-tion with misrouting4 and is thought also to arise from defective melanin synthesis.To investigate whether other conditions than albinism may lead to misrouting andfoveal hypoplasia, we examined three darkly pigmented patients with fovealhypoplasia; one of the patients had Kartagener syndrome.

Patients and methods

Patients

Patient 1 was born in 1985 from healthy, unrelated parents; she has two healthysisters. She always has been dark haired with dark brown eyes, and she tansnormally. From an early age she suffered from recurrent upper respiratory tractinfections. In 1998 she had left sided appendicitis and was subsequently diagnosedwith Kartagener syndrome (McKusick 244400). Kartagener syndrome consists ofprimary ciliary dyskinesia (PCD) with situs inversus totalis. PCD is caused byultrastructural defects of respiratory cilia and sperm tails, leading to recurrentrespiratory tract infections, sinusitis, bronchiectasis, and male subfertility. Thedisorder is autosomal recessively inherited with an incidence between 1/30.000and 1/120.000 live births.5

The patient was regularly ophthalmologically examined from age one, because ofnystagmus; her visual functions and ophthalmological features have remainedstable for twenty years.

Patient 2 and 3 are sisters of Afghan origin, born in 1983 and 1989, respectively.Their parents and siblings are without ocular abnormalities. The patients havealways been dark haired with dark brown eyes and a light brown skin colour. They

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were diagnosed with congenital idiopathic nystagmus in childhood. Their generalhealth is good; they have no situs inversus.

After informed consent, a full ophthalmological examination of the patients wasperformed, including slit lamp examination to detect possible iris transillumina-tion, fundus photography, VEPs, and optical coherence tomography (OCT).

Methods

The angle at which the main temporal arterial branches leave the optic nerve head(ONH) was analysed according to the method described by Neveu2 (Figure 1). InNeveu’s study, the exit angles of normal subjects were 66 ± 11º, for albino patients92 ± 8º.Linear scans of the retina with OCT were performed along four meridians.Standard OCT software was used to generate retinal topographic measurements. VEPs were recorded with five horizontal occipital electrodes referenced to afrontal midline electrode. Misrouting was assessed using pattern onsetstimulation.3 Apart from examining the differential recordings, we assessedmisrouting by calculating the chiasmal coefficient.6 By definition, the chiasmalcoefficient can have a value between -1 and +1; it is negative in misrouting, andpositive in normal subjects. The normative criterion value for our laboratory is M-2SD = -0.17.

Results

Table 1 shows the clinical findings of the patients. All patients had a normally

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RE, right eye; LE, left eye; nyst, nystagmus, α, exit angle of the main temporal arterial branches from the optic nerve head

Table 1. Summary of clinical findings

patient refraction visual acuity nyst anterior segment OCT αOD/OS RE LE upper lower

1 S+2.0=C-2.0x10º/ 0.2 0.2 + dark brown irides, foveal 72º 64ºS+2.5=C-2.0x180º no transillumination hypoplasia



pigmented fundus with no apparent foveal region (Figure 1). The exit angles of thearteries2 lay below the mean minus 2 SD of the angles of the albino group (P<0.05)and were well within the range of the angles in normal subjects.OCT showed extension of all neurosensory retinal layers through the area inwhich the fovea would normally be located7 (Figure 2).The VEP recordings showed a maximal positive CI component over the righthemisphere for the left eye, and over the left hemisphere for the right eye, withthe CI almost absent over the ipsilateral hemispheres (Figure 3). The differentialelectrodes showed opposite polarity for the recordings of the two eyes. Thechiasmal coefficients of all three patients were significantly indicative ofmisrouting.

Discussion

We demonstrated misrouting and foveal hypoplasia in two darkly pigmentedsisters, and in an unrelated female patient with Kartagener syndrome. All patientshad dark brown eyes, no iris transillumination or fundus hypopigmentation, and aretinal vessel pattern different from that found in albinism.

The combination of Kartagener syndrome and ocular abnormalities has not beendescribed before. Because of the wide clinical and genetic variability in Kartagenersyndrome,5 patients may only rarely have misrouting. Alternatively, thecombination of Kartagener syndrome with misrouting may go unnoticed: in ourpatient situs inversus was only diagnosed by coincidence because of a perforatedappendix.

Earlier studies have described female patients with "autosomal recessive ocularalbinism (AROA)", which is now considered to represent clinically mild forms ofoculocutaneous albinism type I or type II,8 resulting from mutations in thetyrosinase or P genes, respectively. AROA seems very unlikely in our patients,because of the absence of any ocular pigmentary abnormality. Furthermore, in ourpatients the exit angles of the retinal blood vessels fell within normal range, anddid not fit the pattern as described in albinism.2

The pathogenesis of misrouting in the three patients is unknown. Because severalgenes have been identified with key roles in regulating optic chiasm development,9

a defect in one of them could possibly lead to misrouting. Some of these genesshow overlap with genes involved in left-right asymmetry.10 Further molecular

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studies are needed to determine which genes are involved in causing Kartagenersyndrome with misrouting.

In addition to misrouting, foveal hypoplasia is a striking feature of our patients. Inalbinos both features are thought to arise because of defective retinal melaninsynthesis.11 However, the combination of both abnormalities in our non-albinoticpatients suggests that there must be a different explanation for their simultaneousoccurrence, for instance foveal hypoplasia causing misrouting, or vice versa.Because it has been shown in aniridia patients that foveal hypoplasia does not leadto misrouting,2 we propose the hypothesis that misrouting may exert a retrogradeinfluence on foveal development.

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Figure 1. Fundus photographs of patient 1 (top left), patient 2 (bottom left), patient 3 (top right), and the perip-heral retina of patient 2 (bottom right). In patient 2, measurement of the exit angle (α) of main temporal arterialbranches to the horizontal meridian is indicated, according to the method described by Neveu.2 The horizontalmeridian bisects the optic nerve head (ONH), the vertical meridian extends from the temporal edge of the ONHand intersects the inferior/superior temporal artery. The angle was measured between the point on the horizontalmeridian where the artery exits the ONH and the point where the vertical meridian intersects the arterial vessel.

The development of the foveal pit occurs from 24 weeks gestation to 45 monthspostpartum.12-13 It has been suggested that critical densities of the central ganglioncell layer (GCL) are required for normal development of the foveal pit.14 Thishypothesis may provide an explanation for the foveal hypoplasia in albinism,because some studies have demonstrated a reduced GCL density in the areacentralis of albino mammals,15 while cone numbers and mosaic are normal.16

However, the reason for this reduced GCL density remains unclear. A correlationhas been suggested between rod numbers and ganglion cell density,16 but such acorrelation does not explain why a reduced GCL density occurs only centrally,while the total number of retinal ganglion cells in adult albino retina is normal.17

Furthermore, there does not seem to exist a correlation between levels of pigmen-tation and deficits in the central ganglion cell layer.18

Because of the findings in our patients, and because foveal pit formation occurs ata time when chiasmal decussation is already established, we propose the hypothesisof a retrograde influence on foveal development.Retrograde influences originate from the visual cortex or optic nerves and tracts.Humans with misrouting due to albinism show reorganization of the normalcortical topographic representation.19 They also have smaller optic nerves, tracts

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Figure 2. OCT of patient 1. There is extension of all neurosensory retinal layers through the area in which the fovea would normally belocated. No clivus, anticlivus, or foveal pit can be seen. Results of the three subjects were similar.

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Figure 3.From top to bottom; responses to pattern onset-offset stimulation of patient 1, patient 2, and patient 3. The dif-ferential electrodes O1 – O2 and T5 – T6 show a positive peak with stimulation of the right eye, which means thatthe response is found in the left hemisphere. For the left eye, this peak is negative, caused by a response of theright hemisphere. All three chiasmal coefficients were significantly indicative of misrouting.

and chiasms than normal subjects.20 Abnormalities of the striate cortex have beenshown to influence developing ganglion cells, with the period of maximalretrograde influence occurring in the first months of life, thus overlapping withthe period of foveal development.21

An additional indication of such a retrograde influence is the finding of anunderdeveloped area centralis in achiasmatic dogs.23 Furthermore, fovealhypoplasia also occurs in cases of severe optic nerve hypoplasia and colobomata.23

The hypothesis also explains why the association of misrouting with fovealhypoplasia is much stronger than with other signs of albinism.4

In conclusion, this study shows for the first time misrouting and foveal hypoplasiain three patients without albinism, and proposes an alternative mechanism for thedevelopment of foveal hypoplasia. Further studies are needed in patients withfoveal hypoplasia caused by a variety of conditions, to understand the preciseassociation between the optic chiasm and foveal development.

Acknowledgements

We like to thank Dr. Mary van Schooneveld for her useful comments, and Ms.Celia de Ruiter for performing the OCTs.

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References

1. Russell-Eggitt I. Albinism. Ophthalmol Clin North Am 2001;14(3):533-46

2. Neveu MM, Holder GE, Sloper JJ, Jeffery G. Optic chiasm formation is independent of foveal

development. Eur J Neurosc 2005;22:1825-1829.


of the visual pathway anomaly. Exp Brain Res 1984;53:285-294.

4. Dorey SE, Neveu MM, Burton LC, Sloper JJ, Holder GE. The clinical features of albinism and

their correlation with visual evoked potentials. Br J Ophthalmol 2003;87:767-772.

5. Geremek M, Witt M. Primary ciliary dyskinesia: genes, candidate genes and chromosomal

regions. J Appl Genet 2004;45:347-361.

6. Jansonius NM, van der Vliet TM, Cornelissen FW, Pott JW, Kooijman AC. A girl without

chiasm: electrophysiologic and MRI evidence for the absence of crossing optic nerve fibers in a

girl with a congenital nystagmus. J Neuroophthalmol 2001;21(1):26-29.

7. Meyer CH, Lapolice DJ, Freedman SF. Foveal hypoplasia in oculocutaneaous albinism

demonstrated by optical coherence tomography. Am J Ophthalmol 2002;133(3):409-410.

8. Morell R, Spritz RA, Ho L, Pierpont J, Guo W, Friedman TB, Asher JH. Apparent digenic

inheritance of Waardenburg syndrome type 2 (WS2) and autosomal recessive ocular albinism

(AROA). Hum Molec Genet 1997;6(5):659-64.

9. Jeffery G, Erskine L. Variations in the architecture and development of the vertebrate optic

chiasm. Prog Retin Eye Res 2005; 24(6):721-53.

10. Elms P, Scurry A, Davies J, Willoughby C, Hacker T, Bogani D, Arkell R. Overlapping and

distinct expression domains of Zic2 and Zic3 during mouse gastrulation. Gene Expr Patterns

2004;4(5):505-11.

11. Jeffery G. The albino retina: an abnormality that provides insight into normal retinal

development. Trends Neurosci 1997;20:165-169.

12. Provis JM, Diaz CM, Dreher B. Ontogeny of primate fovea: a central issue in retinal

development. Prog Neurobiol 1998;54:549-581.

13. Hendrickson AE, Yuodelis C. The morphological development of the human fovea.

Ophthalmology 1984;91(6):603-12.

14. Leventhal AG, Ault SJ, Vitek DJ, Shou T. Extrinsic determinants of retinal ganglion cell

development in primates. J Comp Neurol 1989;286(2):170-89.

15. Jeffery G, Kinsella B. Translaminar deficits in the retinae of albinos. J Comp Neurol

1992;326(4):637-44.

16. Jeffery G, Darling K, Whitmore A. Melanin and the regulation of mammalian photoreceptor

topography. Eur J Neurosci 1994;6(4):657-67.

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17. Rachel RA, Dölen G, Hayes NL, Lu A, Erskine L, Nowakowski RS, Mason CA. Spatiotemporal

features of early neurogenesis differ in wild-type and albino mouse retina. J Neurosc

2002;22(11):4249-4263.

18. Donatien P, Aigner B, Jeffery G. Variations in cell density in the ganglion cell layer of the retina

as a function of ocular pigmentation. Eur J Neurosci 2002;15(10):1597-1602.

19. Hoffmann MB, Tolhurst DJ, Moore AT, Morland AB. Organization of the visual cortex in

human albinism. J Neurosci 2003;23(26):8921-30.

20. Schmitz B, Schaefer T, Krick CM, Reith W, Backens M, Käsmann-Kellner B. Configuration of

the optic chiasm in humans with albinism as revealed by magnetic resonance imaging. Invest

Ophthalmol Vis Sci 2003;44(1):16-21.

21. Herbin M, Boire D, Theoret H, Ptito M. Transneuronal degeneration of retinal ganglion cells

in early hemispherectomized monkeys. Neuroreport 1999;10(7):1447-52.

22. Hogan D, Williams RW. Analysis of the retinas and optic nerves of achiasmatic Belgian

sheepdogs. J Comp Neurol 1995;352(3):367-80.

23. Olsen TW, Summers CG, Knobloch WH. Predicting visual acuity in children with colobomas

involving the optic nerve. J Pediatr Ophthalmol Strabismus 1996;33(1):47-51.

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Chapter 7

Mutations in GRM6 Cause Autosomal Recessive Congenital Stationary NightBlindness with a Distinctive Scotopic 15-Hz Flicker Electroretinogram

Zeitz C1/ van Genderen MM,2 Neidhardt J,1

Luhmann UFO,1 Hoeben FP,2 Forster U,1 Wycisk K,1

Mátyás G,1 Hoyng CB. ,3 Riemslag FCC,2 Meire FM,2

Cremers FPM ,4 and Berger W 1

Invest Ophthalmol Vis Sci 2005;46: 4328 - 4335

Christina Zeitz and Maria M. van Genderen contributed equally to the work and therefore should

be considered equivalent authors.

From the1 Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical

Genetics, University of Zurich, Schwerzenbach, Switzerland; the2 Institute for the Visually

Impaired Bartiméus, Zeist, The Netherlands; and the Departments of 3Ophthalmology and4Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.



Mutations in GRM6 Cause Autosomal Recessive

Congenital Stationary Night Blindness with a

Distinctive Scotopic 15-Hz Flicker Electroretinogram

Purpose: Congenital stationary night blindness (CSNB) is a group ofnonprogressive retinal disorders characterized by impaired nightvision that occurs in autosomal dominant, autosomal recessive, orX-linked forms. Autosomal recessive (ar)CSNB seems to be veryrare. Mice lacking the metabotropic glutamate receptor 6 (Grm6)have a defect in signal transmission from the photoreceptors toON-bipolar cells. In the current study, the human orthologue(Grm6) was screened as a likely candidate for arCSNB.

Methods: arCSNB individuals of five families were screened for mutations inGRM6. Subsequently, they were examined with standard and 15-Hz flicker electroretinography (ERG). These recordings werecompared with those of patients with X-linked CSNB1.

Results: Affected individuals in three of five families carried eithercompound heterozygous or homozygous mutations in Grm6.Strikingly, all of them displayed a distinctive abnormality of the rodpathway signals on scotopic 15-Hz flicker ERG.

Conclusions: The novel profile identified in this study suggests the existence ofmore than two rod pathways. The distinctive ERG feature was notobserved in patients with X-linked CSNB1 and additional affectedindividuals with unknown molecular defect. These observations willhelp to discriminate autosomal recessive from X-linked recessivecases by ERG and molecular genetic analysis.

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Congenital stationary night blindness (CSNB) comprises a group of nonprogres-sive retinal disorders characterized by impaired night vision and sometimes otherocular symptoms such as decreased visual acuity, myopia, hyperopia, andnystagmus. X-linked and autosomal recessive (ar)CSNB are characterized by anelectronegative electroretinogram (ERG) showing normal or near-normal a-waves, but a selective reduction of the b-wave.1 Mutations in two genes, NYX andCACNA1F are responsible for two X-linked types of CSNB designated complete(CSNB1) and incomplete (CSNB2), respectively.2-5

CSNB1 has been associated with absent rod and normal or subnormal cone b-wave amplitudes, whereas CSNB2 exhibits some rod function, but both rod andcone ERG signals are considerably reduced in amplitude. These types can bedistinguished from each other by means of the standard ERG. However, so far,patients with arCSNB cannot be discriminated from those with CSNB1 by clinicalfeatures and standard ERG measurements. The ERG b-wave reflects thetransmission of the signal from the photoreceptors to the second-order neurons.Photoreceptors, when activated by photons, release glutamate as a neurotransmit-ter, which then stimulates either ON or OFF bipolar cells. Although rods synapseonly with rod ON bipolar cells, cones connect with both cone ON and OFFbipolar cells.6 Rod signals in normal human subjects use two different retinalpathways: the rod ON bipolar-amacrine II cell pathway (slow, sensitive rodpathway) and the rod-cone gap junction-cone ON bipolar pathway (fast,insensitive rod pathway). The existence of these two rod pathways has beendemonstrated by means of ERG studies using 15-Hz flicker at scotopic inten-sities.7, 8 At scotopic (starlight) luminance levels, the responses to 15-Hz flicker aredominated by the slow rod pathway and at mesopic (twilight) levels by the fast rodpathway. Between these luminance levels, destructive interference produces apsychophysical flicker null and a minimum ERG amplitude with a 180° phaseshift, because the signals of both pathways are equal in amplitude but opposite inphase. Electrophysiological studies identified the rod ON pathway as the primarysite of the defect in CSNB1.9, 10 The ON bipolar dendrites express mainly the high-affinity, sixth subtype metabotropic glutamate receptor (mGluR6).11-13 Thephenotype of homozygous knockout mice lacking the gene (Grm6) encodingreceptor mGluR6 resembles that of the human CSNB: a greatly reduced b-waveunder dark-adapted conditions, whereas the a-wave, representing thephotoreceptor activity, is normal. Furthermore, the retinal organization in thesemice shows no obvious changes on the cellular level.14 Because the glutamatereceptor plays a critical role in both rod pathways, mutations in this gene may

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influence both the slow and the fast rod pathway signals. The absence of fundusabnormalities and the electronegative standard ERG renders this gene anattractive candidate for arCSNB. Indeed, most recently, another group identifiedGrm6 mutations in three patients with arCSNB.15 Herein, we report on ERGresponses and Grm6 mutation analysis in seven patients with diagnosed arCSNB.

Subjects and methods

Patients

Seven patients from five unrelated families had congenital stationary nightblindness (CSNB) diagnosed by means of a complete ophthalmic examination,including funduscopy, dark-adapted thresholds and Ganzfeld ERG, according tothe standard ISCEV (International Society for Clinical Electrophysiology ofVision) ERG protocol.16 The protocol of the study adhered to the tenets of theDeclaration of Helsinki. In two male patients, no pathogenic variant was found inNYX or CACNA1F;17 the other five patients were female offspring of unaffectedparents. The clinical characteristics of the patients are shown in Table 1, thestandard ERGs of the father of the three affected siblings and one of these siblingsare represented in Figure 1 together with the responses of a patient with CSNB1.

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Patient 21973 had Down syndrome, which also may be associated with nystagmus, high myopia, and low visualacuity. Nyst, nystagmus. * Spherical equivalent.

Table 1. Clinical characteristics of the patients with presumed arCSNB

Visual acuity Refractive error* Dark adapted thresholdelevation (log units)

Patient Sex Age (y) OD OS Nyst OD OS21974 F 13 1.0 0.2 - S-9.0 S-10.0 321973 F 10 0.1 0.1 + S-10.0 S-8.0 Not done26162 F 8 0.5 0.5 - S-6.5 S-6.0 326151 F 15 0.2 0.3 + S-3.5 S-2.5 326111 F 11 0.8 0.4 - S-14.0 S-13.5 32496 M 36 0.4 0.4 + S-3.0 S-3.0 2.52445 M 46 0.3 0.1 + S+3.5 S+1.5 2.5

Mutation Analysis

All subjects or their parents gave written informed consent before moleculargenetic testing. Genomic DNA was isolated from EBV-transformedlymphoblastoid cells or blood by standard techniques. The 10 coding exons ofGrm6 were amplified with intronic or exonic primers (Supplementary Table S1,http://www.iovs.org/cgi/content/full/46/ 11/4328/DC1).

Eleven fragments containing the 10 coding exons of Grm6 were amplified withpolymerase (HotFirePoly DNA Polymerase; Solis Biodyne, Tartu, Estonia), 1.5mM MgCl2, and Q solution (Qiagen, Hombrechtikon, Switzerland). The totalvolume of the PCR reaction was 25 µL, and 0.8 µL of the PCR product wastreated with 5 µL of 1:50 diluted clean-up solution (ExoSAP-IT; USB, Cleveland,OH) at 37°C for 15 minutes, to remove unconsumed primers and dNTPs. Aftertreatment, the enzyme was inactivated by heating to 80°C for 15 minutes. After ashort centrifugation step, 1 µL dye-termination and 1.5 µL5 dye-termination buf-fer (BigDye Terminator ver. 1.0; Applied Biosystems, Rotkreuz, Switzerland), 2µL5 Q solution, and 0.8 µL primer (10 pM/µL) were added to each reaction, andstandard sequencing was performed. Sequences were analyzed on an automatedDNA sequencer (Prism 3100; Applied Biosystems).

ERG Stimulation, Recording, and Analysis

The eyes of the subjects were anesthetized with oxybuprocaine 0.4%, and thepupils were dilated with tropicamide 0.5%. Subsequently, DTL electrodes werepositioned on top of the lower eyelid, touching the eye. Pupil diameters weredetermined immediately after the ERG recordings were finished (8–9 mm in allsubjects). For the standard ISCEV ERG measurements, Xenon tube flashes(duration 10 s) were delivered in a custom-made Ganzfeld dome, at one flash every2 seconds for the low (2.6 ND), and one flash every 5 seconds for the standardISCEV intensity (mixed response). Subjects were then light adapted for 10minutes by exposure to a white 30-cd/m2 rod-saturating background, and photopicERGs were recorded to standard ISCEV intensity and to white 30 flashes persecond.

Preamplification (5.000x) and sampling (1000 Hz) plus multiplexing wereperformed with a custom-made, battery-driven preamplifier unit. These multi-plexed signals were optically transmitted to the recording computer. Signals weredigitally filtered online, using a bandpass filter from 0.5 to 200 Hz (3 dB). Sweep

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length for averaging was 200 ms, with an additional 40-ms prestimulus period.During the averaging process, the raw ERG sweeps were displayed for exami-nation and judged for inclusion or exclusion by the examiner. This process resultsin a better artifact rejection than is usually obtained with level rejection. Only theaveraged signals and the plus–minus averages18, 19 were stored for further analysis.ERG responses to 15-Hz flicker were recorded in six of the seven patients witharCSNB. Because patient 21973 had Down syndrome, she was unable to complywith the lengthy 15-Hz procedure. The 15-Hz protocol was recorded intermixedwith the standard ISCEV ERG at the appropriate intensities. Xenon flash (10µs)intensities of 15 flashes per second (sweep length, 135 ms, 500–1000 averages)were attenuated by switching between 0.3 and 0.6 J (Nihon Kohden, Tokyo,Japan) and inserting neutral density (ND) filters (Schott, New York, NY) mountedin a filter wheel. Using maximum attenuation, we obtained a stimulus intensity of1.56 log scot-td/s, reaching 0.44 log scot-td/s in nine steps. The 15-Hz responses were trend corrected off-line and filtered by a movingaverage filter with a rectangular window of 20 ms (21 data points). The 15-Hz

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Figure 1. ISCEV Standard ERG of the father 26163, a patient with CSNB1, and one with arCSNB (patient21974). Top to bottom: photopic response, 30-Hz photopic response, scotopic response, mixed response, oscillato-ry potentials (Gaussian window moving average filter, σ=1.5 ms). The responses of the father could not be dis-tinguished from our normative data (N=67). In the subject with CSNB1 and the one with arCSNB, there was norecordable scotopic response, and the mixed response featured a minimal b-wave and showed the characteristicelectronegative waveform. A remnant of a single oscillatory potential was found at approximately 33 ms, with anamplitude within normal limits. The photopic response showed a characteristic broad a-wave. The b-waves of thephotopic responses were within normal limits in both patient groups. The 30-Hz photopic responses also had nor-mal amplitudes and implicit times in both patient groups.

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scotopic flicker ERG in normal subjects showed a minimum response atapproximately 0.56 log scot-td/s. The ERG signals to flicker intensities between1.56 and 0.72 log scot-td/s were therefore considered to be dominated by slow rodERG signals. 20 Amplitudes and phases of the 15-Hz averaged amplitudes and theplus-minus–averaged signals18, 19 were obtained by off-line Fourier analysis.Amplitudes were considered significant if the ratio of the amplitudes of theaverage and the plus–minus average was above 3 (P< 0.01). For those responses,we plotted phases according to a strict criteria, assuming that phases decreasesmonotonously with stimulus intensity. We subtracted an integer multiple of 360°from the calculated phase (phase is indeterminate for multiples of 360°). An extra360° was subtracted whenever the phase for that intensity seemed to increase 1 SDof the phase in unaffected subjects. Apart from the patients with arCSNB, six patients with CSNB1 (age, 4-14 years)were tested with the 15-Hz protocol. The CSNB1 diagnosis was based on apositive X-linked family history in three consecutive generations, clinicalexamination, standard ISCEV ERGs, and 30-Hz photopic flicker ERGs.21

Results

Mutation Analysis

The GRM6 gene consists of 10 exons and encodes a protein with 878 amino acids.We performed mutation analyses in the open reading frame (ORF) of the 10 exonsand parts of the flanking introns of GRM6 (Accession NM 000843; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Centerfor Biotechnology Information, Bethesda, MD) in five unrelated families, with atleast one member displaying lack of nocturnal vision and an electronegative ERG.

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Figure 2. Mutations in GRM6 cause autosomal recessive CSNB. Direct sequencing of the 10 coding exons of theGRM6 gene revealed five different mutations in three families with arCSNB. (A) Pedigree for arCSNB-bearing families 1, 2, and 3. Circles: females; squares: males; open symbols: unaffected;semifilled symbols: carrier; and filled symbols: affected. Patient identification numbers are indicated above thesymbols, mutations below. The father in family 2 was not available for testing. (B) Electropherograms showing the five different mutations. Arrows: site of the mutation; star: common poly-morphisms in the investigated sequences. Patient 2445 of family 1 had two compound heterozygous mutations:one missense mutation (c.137C→T) in exon 1 and one nucleotide insertion (c.720_721insG) in exon 3. The mot-her of this patient was heterozygous for the insertion, whereas the father carried the missense mutation. The unaf-fected sister of patient 2445 showed none of the alterations. In family 2, the male patient, 2496 revealed a homo-zygous missense mutation (c.1565G→A) in exon 8. The mother and the unaffected brother were heterozygous forthis mutation, whereas the unaffected sister did not show the mutation. In family 3, the three affected siblings26162, 21973, and 21974 carried a heterozygous (c.172G→C) and 19-nucleotide duplication (c.57_75dupl19).Both mutations were found in exon 1. The mother was also heterozygous for the duplication, whereas the fathercarries the p.Gly58Arg exchange.

In patient 2445 of family 1, we identified two compound heterozygous mutations:one missense mutation (c. 137C→T) leading to a Pro46Leu change in exon 1 andone nucleotide insertion (c.720_721insG) in exon 3, leading to a frame shift atposition 243 and a premature stop codon 39 amino acids later. The mother of thispatient was heterozygous for the insertion, and the missense mutation wastransmitted from the father. The unaffected sister of patient 2445 showed none ofthe alterations (Fig. 2). In family 2, a male patient (2496) carried a homozygousmissense mutation (c. 1565G→A), leading to a p.Cys522Tyr change in exon 8 ofGRM6. The mother and the unaffected brother were heterozygous for thismutation, whereas the unaffected sister was homozygous wild type. The father wasnot available for testing (Fig. 2). In family 3, the three affected sisters (26162,21973, and 21974) carried a heterozygous duplication of 19 nucleotides(c.57_75dupl19) in exon 1, which denotes a frame-shifting exchange of Leucine(Leu) at position 26, as the first affected amino acid, with valine (Val), creating anew ORF ending in a stop at position 168. A second mutation (c.172G→C) wasidentified in the same exon and leads to a p.Gly58Arg substitution. The motherwas heterozygous for the duplication, whereas the father carried the p.Gly58Argexchange (Fig. 2). None of the mutations was detected in more than 100 healthycontrol chromosomes of European descent. Two of the seven analyzed patients(26111, 26151) did not show a mutation in the complete ORF of GRM6. Patient26111 had one unaffected sister and one unaffected brother. Patient 26151 hadone unaffected sister. The parents of both patients, as well as their siblings wereunaffected, as were the grandparents of patient 26151 (data not shown).

Protein Analysis

mGluR6 is a metabotropic glutamate receptor that belongs to a G-protein-coupled receptor family that has been divided into three groups based on theirhomology, putative signal transduction mechanisms, and pharmacologicproperties.22 mGluR1 and mGluR5 belong to group I and activate phospholipaseC. mGluR2 and mGluR3 belong to group II, and group III includes mGluR4,mGluR6, mGluR7, and mGluR8. Group II and III receptors are involved in theinhibition of the cyclic AMP cascade, but differ in their agonist. A multiplesequence alignment of all metabotropic glutamate receptors of group III revealedthat all missense mutations (p.Pro46Leu, p.Gly58Arg, and p.Cys522Tyr) arelocated in residues that are conserved among the other glutamate receptors of thisgroup. In addition, the positions of the p.Gly58Arg and the p.Cys522Tyrexchanges are shared by all eight metabotropic glutamate receptors (data not

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shown). Comparative sequence analysis of mGluR6 and the crystallized ratmGluR1 revealed that the p.Pro46Leu and the p.Gly58Arg exchanges are locatedin the first ligand binding domain, whereas the p.Cys522Tyr exchange is predictedto be in a highly conserved cysteine-rich region. The two mutations leading toframe shifts (p.Leu26fs and p.Val243) occur shortly before the first and within thesecond ligandbinding domain, respectively (Figs. 3A, 3B).

Slow and Fast Rod ERG Signals

Patients in whom CSNB was diagnosed by means of standard ERG measurements(see the Methods section) were further analyzed with a 15-Hz scotopic ERG totest for a clinical correlation with the identified mutations in GRM6. This methodwas used because it allows discrimination between the two rod pathways: the rodON bipolar-amacrine II cell pathway (slow) and the rod-cone gap junction-coneON bipolar pathway (fast), and both use mGluR6 to signal.

The ERG responses to 15-Hz flashes at scotopic conditions in a normal subject, apatient with clinically diagnosed CSNB1, and two with arCSNB, without and withthe GRM6 mutation, are shown in Figure 4. In the normal subject, the amplitudeof the ERG signal increased slightly with increasing flicker intensity from 1.56 to1.26 log scot-td/s and decreased thereafter. There was a minimum ERG responseat flicker intensities between 0.72 and 0.42 log scot-td/s. The phase of the ERGresponses abruptly increased by approximately 180° (corresponding to a half-cycle) as the amplitude minimum was crossed, consistent with destructive inter-ference between the slow and fast rod ERG signals being the cause of theamplitude minimum. The ERG signals rapidly increased in amplitude at higherflicker intensities (from 0.42 log scot-td/s onward). In the patient with CSNB1,the ERG signals of the slow rod pathway at flicker intensity between 1.56 and 0.56log scot-td/s were indistinguishable from noise. An ERG signal became apparentat the higher flicker intensities. However, this signal was reduced in amplitude.The 15-Hz scotopic responses were similar in all six tested patients with CSNB1.

In patient 21974 of family 3, the amplitude of the ERG response showed acomplicated profile: a large response at the lowest intensity and a minimal respon-se at four intensities (1.42, 0.72, 0.26, and 0.44 log scot-td/s). The respectiveresponses of all our examined patients with GRM6 mutations (2445, 2496, and26162) had similar characteristics. This behavior of the slow rod pathway signalscould not be demonstrated in the two other patients presumed to have arCSNB

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Figure 3. GRM6 mutations in the different regions of the glutamate receptor. The relative positions of thedomains were predicted among the crystal structure of the rat Grm1. (A) The ligand-binding domains 1 (LB1; fil-led boxes) and 2 (LB2; dotted boxes) are shown. Unfilled boxes: protein regions not determined by crystallization;unfilled arrows: missense mutations; black arrows: frameshift mutations. The p.Pro46Leu and the p.Gly58Argexchanges are located in the first ligand-binding domain, whereas the p.Cys522Tyr exchange is predicted to be ina highly conserved cysteine rich region. One frame shift identified in this study starts shortly before LB1(Leu26fs), whereas the other is located in LB2 (Valfs243). (B) Schematic representation of the mGluR6 proteinillustrating the extracellular bilobed glutamate-binding domains (LB1 and LB2) connected by a cysteine-richregion to the transmembrane domain with an intracellular C terminus. The circular object represents the ligandglutamate.

(26111 and 26151) without GRM6 mutation nor in the patients with CSNB1analyzed. 20 For the arCSNB patients with GRM6 mutations consistent responseswere found for most of the stimuli in the lower range, although some amplitudeswere below the normal mean (Fig. 5A). For these patients the corresponding phaseplot is given in Figure 5B. The patients showed a much more rapid phase decreaseat the lower intensity range than in unaffected subjects. Finally, in one of thearCSNB subjects an extra phase shift occurred around 0 log scot-td/s. In theCSNB1 group the responses at stimulus intensities below 1.0 log scot-td/s werenot significant. From 0.72 to 0.26 log scot-td/s some subjects showed significantresponses at some intensities; however, with larger interindividual variability. Onlyat intensities of 0.28 log scot-td/s and higher, response amplitudes were syste-matically growing. We did not make a phase plot for the patients with CSNB1,because they had so few significant signals at the lower intensities that phasescould easily vary 360°.

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Figure 4. Rod ERG responses to 15-Hz flicker stimulation obtained from a normal subject, a patient withCSNB1 and two with arCSNB (patient 26151 without and patient 21974 with a GRM6 mutation). Stimulus inten-sity was 1.56 log scot-td/s at maximum attenuation, reaching 0.44 log scot-td/s in nine steps. ERG signals to flic-ker intensities between 1.56 and 0.72 log scot-td/s were considered to represent primarily the slow rod pathway.In the patients with arCSNB without mutations in GRM6, the slow rod pathway signals could not be distinguis-hed from noise, whereas in patients with GRM6 mutations the signals had large amplitudes and several reversingminima. In patient 21974, the responses of the right eye are plotted in gray, and those of the left eye in black.

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Figure 5. (A) Amplitudes of the 15-Hz component for the unaffected subjects (n 10; filled symbols; bars SD,interindividual). Shaded symbols: amplitudes in the four patients with arCSNB with GRM6 mutations: 2496 S,2445 R, 21974 E, and 2162 P. Only significant responses were plotted (S-N ratio 3, P<0.01). Below 0.42 log scot-td/s most of the responses of the GRM6 subjects had an S-N ratio above 3. (B) Phases for the 15-Hz components in the unaffected subjects (filled symbols; bars SD, interindividual) and thepatients with arCSNB with GRM6 mutations (shaded symbols). At intensities below 1 log scot-td/s, all patientsshowed an extra phase shift, apart from the phase shift at the intensity range at which unaffected subjects alsoshowed a shift.

Discussion

With a candidate gene approach, we identified, in three of five families withdiagnosed CSNB, mutations on both alleles of the GRM6 gene. This gene was anattractive candidate because a mouse model lacking the metabotropic glutamatereceptor 6 (GRM6) showed a defect in the transmission of the light signal from thephotoreceptors to the ON-bipolar cells, similar as observed in patients with X-linked or arCSNB. Sequence analysis verified our hypothesis. One patient showeda homozygous missense mutation, whereas the others were compoundheterozygous. The latter carried different missense mutations on one allele andinsertions on the second. All missense mutations affected amino acid residues thatare conserved among the other glutamate receptors of group III. In addition, fourof the five different mutations occurred in the first part of the receptor, in exons 1and 3. The third missense mutation affects one of several conserved cysteineresidues that are thought to form disulfide bridges outside the cell membrane. Ourfindings are supported by a very recent report in which also missense mutations inGRM6 were associated with arCSNB.15 The x-ray crystal structure of the ratmGluR1 showed two conformations that undergo a dynamic change between theopen and the closed form. On agonist binding the closed conformation isstabilized and thereby activates the receptor. Within the three-dimensionalstructure of mGluR6, the position of the missense mutations identified in ourstudy is close to the binding region of glutamate (data not shown). Although thedirect binding pocket is not affected, our findings suggest that the mutated regionshave a role in recruitment or binding of glutamate. This notion is supported byreports showing that amino acids from the upper lobe are predominantly involvedin anchoring the ligand and that polarity of these residues is conserved among thedifferent metabotropic glutamate receptor subtypes 23 Furthermore, site-directedmutation analyses have shown that in vitro polar or charged amino acids from theupper lobe cleft region of the molecule are key determinants of agonist affinity.24,

25 One of the missense mutations reported by Dryja et al15 was also identified in theligand-binding domain, whereas another one is located in the intracellular loopbetween two transmembrane domains. Two other mutations, found in exon 8, leadto protein truncation. Together, these findings suggest that the identifiedmutations lead to the loss of function of the glutamate receptor. Future studies willshow how these mutations exactly modulate the functional properties of thereceptor.

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With ophthalmic examinations, including standard ERGs, one cannot distinguisharCSNB from CSNB1 in patients. However, rod pathway responses to 15-Hzflashes showed a distinctive profile in patients with mutations in GRM6, differentfrom those of patients without GRM6 mutations and also different from patientswith CSNB1. Scholl et al 20 demonstrated minimal slow rod pathway signals inpatients with CSNB1 and reduced fast rod signals with increased phases, by using15-Hz scotopic ERG measurements. They suggested that defective nyctalopin inpatients with CSNB1 leads to a complete blockage of signal transmission withinthe ON pathway. This hypothesis was recently further supported by investigationsin primates.9 In accordance with Scholl’s research, our study has also demonstratedminimal slow rod pathway signals in patients with CSNB1 and reduced fast rodsignals with increased phases (Fig. 4).

In contrast, the 15-Hz scotopic signals in the patients with arCSNB with GRM6mutations had remarkable abnormal phase behavior, with several minimumresponses. In Scholl’s study of patients with CSNB1, abnormal phase behavior wastaken as proof of absence of responses at the lower flicker intensities, because in nonaffected persons, there is a proper alignment of the phases betweenadjacent ERG responses. In CSNB1, the slow rod pathway signals are very small,and therefore their phases may indeed be substantially influenced by noise.However, in the patients with arCSNB carrying GRM6 mutations the amplitudesof the responses were significantly above the noise level, and the phases wereconsistent between both eyes of the same patient. Because similar phase shiftsoccurred in all patients with GRM6 mutations, this abnormal phase behavior maybe a characteristic feature of this retinal disorder.

Large phase shifts between adjacent ERG responses may be caused by abnormalmodes of transmission within the rod ON bipolar pathway, but several minima inthe amplitude versus intensity plot can only be explained by interference betweenat least two signals. The first minimum response is apparent at -1.42 log scot-td/s.At this intensity, the largest signals in nonaffected persons are those of the slow rodpathway. To destructively interfere with the slow pathway, fast rod pathway signalsshould have approximately similar amplitudes. However, at this very low intensity,the fast signals have been shown to be much smaller than the slow rod pathwaysignals.26 Previous studies showed that significant interference of slow rod pathwaysignals with cone signals is also unlikely. 7, 27

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The interference pattern in the patients with arCSNB therefore raises thequestion of the existence of more than two rod pathways, an assumption that wasalso previously suggested.28, 29 The slow rod signal may consist of more than onemode of transmission near absolute threshold, as has been demonstrated in cats.28,30

Apart from mGluR6, ON bipolar dendrites also bear ionotropic glutamatereceptor subunits 31 A second slow rod pathway may consist of signals traveling viathese receptors. In normal subjects, the interference pattern of two slow rodtransmission modes could result in the normal overall slow pathway signal. If aGRM6 mutation leads to abnormal functioning of one of those routes, aninterference pattern, as found in our patients, may be conceivable.

Alternatively, rod signals may be transmitted via a direct rod to OFF cone bipolarpathway. This pathway exists in rodents and cats and may be a common feature ofmammalian retinas.31–34 In a mouse retina genetically modified to be coneless(mimicking human achromatopsia), direct signals via rods to OFF cone bipolarcells have been demonstrated.32 The residual fast rod signal in CSNB1 could be theresult of rod signal transmission through rod–cone gap junctions to OFF conebipolar cells.9 This third pathway uses the glutamate receptor iGlur2 instead of themGlur6 transmission of the ON pathways.34 It is supposed to be less sensitive thanthe slow rod pathway. 28, 32 Consequently, signals of this pathway at -1.42 log scot-td/s may be very small, as for the fast rod pathway signals. However, with phaseshifts of either the fast rod pathway signals or the third rod pathway signals, orboth, interaction could result in a larger combined signal. This could, in turn,destructively interfere with the slow pathway signals.

In patients with CSNB1, absence or near absence of slow rod pathway responsesresults in an undetectable rod mediated response in the standard ERG and anabsent rod component in dark adaptation. The slow rod pathway responses in thepatients with arCSNB with GRM6 mutations seem to indicate that not onlyamplitude, but also timing of the signals is essential in generating a standard ERGrod response and perception of the stimulus. Proper alignment of consecutiveresponses appears to be necessary to create an overall signal.

In a recent study by Dryja et al,15 three patients carrying GRM6 mutations showedmarkedly reduced ON responses but nearly normal OFF responses to a sawtoothflickering light under photopic conditions. The authors suggest the possibility of

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alternative routes for rod signaling, because the visual functions of these patientscould not be fully explained by defective signaling in the ON bipolar pathway. Therod pathway profile found in our study may be the first direct evidence of theexistence of such alternative pathways in humans. However, the number ofpatients in our study was small, due to the rarity of arCSNB. Larger numbers ofpatients with GRM6 mutations are needed to substantiate our results, andadditional experiments, for instance with different scotopic flicker frequencies, arenecessary to clarify further the abnormal phase behavior of the slow rod pathwaysignals. It would also be interesting to see whether other not yet identified genedefects will result in a similar phenotype. Further examinations are necessary toclarify the abnormal phase behavior of the slow rod pathway signals.

In summary, these studies show that mutations in GRM6 can lead to one form ofarCSNB in patients with characteristic 15-Hz flicker ERG responses. This findingmay imply the existence of more than the two reported rod pathways for signaltransduction from photoreceptors to second-order neurons in the mammalianretina. Genes encoding other components involved in this pathway may provideadditional candidates for arCSNB.

Acknowledgments

The authors thank the patients and their families for participation in this study andBarbara Kloeckener, Oscar Estevez, and Dick van Norren for critical commentson the manuscript.

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1. Miyake Y, Yagasaki K, Horiguchi M, et al. Congenital stationary night blindness with negative

electroretinogram: a new classification. Arch Ophthalmol. 1986;104:1013–1020.

2. Bech-Hansen NT, Naylor MJ, Maybaum TA, et al. Loss-of-function mutations in a calcium-

channel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night

blindness. Nat Genet. 1998;19:264–267.

3. Strom TM, Nyakatura G, Apfelstedt-Sylla E, et al. An L-type calciumchannel gene mutated in

incomplete X-linked congenital stationary night blindness. Nat Genet. 1998;19:260–263.

4. Bech-Hansen NT, Naylor MJ, Maybaum TA, et al. Mutations in NYX, encoding the leucine-

rich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness.

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5. Pusch CM, Zeitz C, Brandau O, et al. The complete form of X-linked congenital stationary

night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein. Nat

Genet. 2000;26:324–327.

6. Sieving PA. Photopic ON- and OFF-pathway abnormalities in retinal dystrophies. Trans Am

Ophthalmol Soc. 1993;91:701–773.

7. Sharpe LT, Fach CC, Stockman A. The spectral properties of the two rod pathways. Vision Res.

1993;33:2705–2720.

8. Sharpe LT, Stockman A, MacLeod DI. Rod flicker perception: scotopic duality, phase lags and

destructive interference. Vision Res. 1989;29:1539–1559.

9. Khan NW, Kondo M, Hiriyanna KT, et al. Primate retinal signaling pathways: suppressing ON-

pathway activity in monkey with glutamate analogs mimics human genetic CSNB1-NYX night

blindness. J Neurophysiol. 2005;93:481–492.

10. Langrova H, Gamer D, Friedburg C, et al. Abnormalities of the long flash ERG in congenital

stationary night blindness of the SchubertBornschein type. Vision Res. 2002;42:1475–1483.

11. Nakajima Y, Iwakabe H, Akazawa C, et al. Molecular characterization of a novel retinal

metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4-

phosphonobutyrate. J Biol Chem. 1993;268:11868–11873.

12. Nomura A, Shigemoto R, Nakamura Y, et al. Developmentally regulated postsynaptic

localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell. 1994;77:361–369.

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localization of the gene for human metabotropic glutamate receptor subtype 6. Eur J Neurosci.

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14. Masu M, Iwakabe H, Tagawa Y, et al. Specific deficit of the ON response in visual transmission

by targeted disruption of the mGluR6 gene. Cell. 1995;80:757–765.

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15. Dryja TP, McGee TL, Berson EL, et al. Night blindness and abnormal cone electroretinogram

ON responses in patients with mutations in the GRM6 gene encoding mGluR6. Proc Natl Acad

Sci USA. 2005;102:4884–4889.

16. Marmor MF, Zrenner E. Standard for clinical electroretinography (1999 update). International

Society for Clinical Electrophysiology of Vision. Doc Ophthalmol. 1998;97:143–156.

17. Zeitz C, Minotti R, Feil S, et al. Novel mutations in CACNA1F and NYX in Dutch families with

X-linked congenital stationary night blindness. Mol Vis. 2005;11:179–183.

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linked complete stationary night blindness carrying mutations in the NYX gene. Invest

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congenital stationary night blindness. Invest Ophthalmol Vis Sci. 1987;28:1816–1823.

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Ophthalmol. 1994;3:433–439.

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Chapter 8

Summary



Summary

Inherited retinal disorders and posterior visual pathway abnormalities areimportant causes of visual impairment in children. Visual electrophysiology oftenis indispensable in diagnosing these conditions.This thesis shows the wide rangeof use of pediatric electrophysiology, and demonstrates its value in establishingdiagnoses, explaining pathophysiological mechanisms, predicting the course of adisease, and supporting new hypotheses.

Chapter 2 describes the results of a retrospective study of 340 electrophysiologicalinvestigations in children, performed over a period of three years. Examinationswere performed without sedation or aneasthesia. Several measures were taken tooptimize compliance and to increase the chance of a successful outcome. In ERGs,these measures consisted of the use of DTL-electrodes, on-line selection ofresponses, and measuring an extended ISCEV protocol. The children had a meanage of 7 ± 5 years; only 4% of the examinations failed because of lack of com-pliance. The most frequently established retinal disorders were progressivetapetoretinal dystrophies, congenital stationary night blindness, and conedystrophy. In VEP testing, misrouting was routinely established in every patient,which resulted in finding unsuspected misrouting in six children. In 26 children(9%) a previously established diagnosis was changed; in 20 of them leading to achange of prognosis. These diagnostic changes profoundly influenced therehabilitation process.

Chapter 3 reports on the ophthalmological and electrophysiological findings in a15-year-old girl with a multi-system disorder. She was deaf, suffered from diabetesmellitus, and had severe anemia requiring blood transfusions every two months.An ERG was performed because of retinal abnormalities. Rod responses weregreatly reduced and cone responses were nearly absent, proving cone-rod dystro-phy. The ocular findings combined with the systemic features led to the diagnosisof thiamine responsive megaloblastic anemia syndrome (TRMA). Patients withTRMA have a defect in active thiamine uptake, leading to damage of varioustissues, including the auditory pathways, the pancreas, and the hematopoieticsystem. Retinal dystrophy was reported in a small number of cases. TRMApatients can be treated with pharmacological concentrations of thiamine, becausethe passive uptake of thiamine is not impaired. Treatment of the girl resulted in

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normalization of her blood cell count and hemoglobin concentration withoutfurther need for blood transfusions.

Chapter 4 describes the ophthalmological and electrophysiological findings in 24patients with Rubinstein-Taybi syndrome, a mental retardation-multiplecongenital anomalies syndrome. Review of the literature showed a wide range ofreported eye abnormalities, predominantly of the external eye, anterior segment,and lens. In contrast, 78% of the 24 investigated patients proved to have a retinaldysfunction; this high frequency had not been described before. Abnormal ERGsshowed a decreased cone response; abnormal pattern onset VEPs lacked a striatecomponent, indicative of macular dysfunction. Four patients had normal ERGsand VEPs, indicating phenotypic, and possibly also genetic, heterogeneity. The retinal and electrophysiological abnormalities occurred more frequently withage, suggesting a progressive retinal dystrophy.

In Chapter 5 the clinical and ophthalmological characteristics of a girl and heryounger brother with ectrodactyly, ectodermal dysplasia, and macular dystrophy(EEM syndrome) are presented. The siblings were the thirteenth and fourteenthpatient with EEM to be described in 49 years, and the first ones in which thediagnosis was established in infancy. When we saw them for the first time, at ageone and seven months, respectively, they already had extensive retinalabnormalities indicative of a congenital retinal dysplasia. Funduscopic appearancehardly changed over a period of ten years, and ERGs remained normal, indicatinga stationary condition rather than a macular dystrophy. The fundus abnormalitiesshowed similarities with the fundus appearance found in severe pseudoxanthomaelasticum, and may therefore be associated with Bruch’s membrane pathology.EEM is caused by mutations in CDH3, encoding P-cadherin; mutations in thisgene are also responsible for hypotrichosis with juvenile macular dystrophy(HJMD).The ocular characteristics of most EEM patients, including ours, seemdifferent from those described in HJMD, but because some patients with HJMD-like macular dystrophy had minimal hand and teeth abnormalities, EEM andHJMD may represent phenotypic heterogeneity of the same syndrome.

In Chapter 6, three female patients are described with misrouting and fovealhypoplasia. One of the patients had Kartagener syndrome, in which the positio-ning of internal organs is inverted (situs inversus). Misrouting is considered to bethe hallmark of albinism, and foveal hypoplasia an important ocular feature of the

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condition. However, pigmentation and retinal vessel patterns of the patientsdisproved albinism. The findings indicated that pigmentation is not the onlydecisive factor in determining misrouting and foveal hypoplasia. Misrouting inthese patients may have been caused by defective genes involved in chiasmdevelopment. Furthermore, we proposed the hypothesis of misrouting exerting aretrograde influence on foveal development, because the study showed thatmisrouting and foveal hypoplasia occur together in different syndromes.

In Chapter 7, the psychophysical, electrophysiological, and genetic characteristicsof autosomal recessive congenital stationary night blindness (arCSNB) aredescribed. The clinical characteristics and the ISCEV standard ERGs of thepatients were indistinguishable from those found in X-linked CSNB1 ("complete"CSNB). Mutation analysis in five of the seven patients showed mutations inGRM6. GRM6 encodes the metabotropic glutamate receptor mGlur6, which is themain receptor of ON bipolar dendrites. Rod signals in humans use two differentrod pathways: the slow rod pathway, from rods to rod ON bipolars, and the fastrod pathway, from rods to cone ON bipolars by way of rod-cone gap junctions.These two pathways can be demonstrated by ERG measurements using 15 Hzflicker at scotopic intensities. With absent mGlur6 we would expect the signals ofboth ON pathways to be absent or nearly absent. However, slow rod pathwaysignals in the patients were clearly recordable, but with an unusual phasebehaviour with several reversal minima. The results indicated the existence ofalternative pathways for rod signalling under scotopic conditions. The unusual 15Hz scotopic measurements were only found in arCSNB patients with GRM6mutations; arCSNB patients without mutations and CSNB1 patients had nonrecordable slow rod pathway signals. 15 Hz scotopic flicker measurements maytherefore be used to distinguish these conditions.

Electrophysiology and rehabilitation; thoughts on future research

In 2001, the World Health Organization (WHO) drafted the InternationalClassification of Functioning, Disability and Health (ICF). This classification wasfirst created in 1980 and then called the International Classification ofImpairments, Disabilities, and Handicaps, or ICIDH. In contrast to ICIDH, thelanguage of ICF is neutral as to etiology, placing the emphasis on function ratherthen condition or disease. The ICF is structured around the following broadcomponents:

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- body functions and structure- activities (related to tasks and actions by an individual) and participation

(involvement in a life situation)- additional information on severity and environmental factors.

Because our electrophysiology department is part of a rehabilitation centre, itshould not only have a function in assessing "body functions and structure", butalso be influential on the other ICF domains: activities, participation, andenvironment. Some examples have already been given in this thesis. In Chapter 2,the consequences of knowing the prognosis of a disorder on education and choiceof career were discussed. Some of the Rubinstein-Taybi patients described inChapter 4 were shown to have a visual impairment with consequences for theirdaily life.

However, during the investigations decribed in this thesis we encountered severalnew questions that need to be elucidated in the future.

1. In Chapter 7, we investigated patients with different forms of congenitalstationary night blindness (CSNB). We noticed that the relationship betweenelectrophysiological results, psychophysical examinations, and problems withnight vision in several types of CSNB and tapetoretinal dystrophies (TRD) is nota simple one. Patients with "complete" CSNB (CSNB1) may experience hardlyany problems with night vision, despite a completely absent rod component intheir dark adaptation curve. Patients with "incomplete" CSNB (CSNB2) oftenhave more complaints related to cone function, like poor visual acuity andphotophobia, than to rod function (this makes the expression "incomplete" CSNBan unfortunate one, because parents tend to think that "incomplete" is a lessserious condition than "complete"). However, in both diagnoses patients withmore severely elevated dark adapted thresholds may experience less problems inthe dark than patients with less abnormal thresholds. Patients with TRD oftenhave serious night vision problems already early in the course of the disease, butthis also tends to vary among different patients. Another interesting condition is arare form of recessive CSNB, with electrophysiologically absent rod (a nor b wave)but completely normal cone function. We propose to investigate these patientgroups electrophysiologically and psychophysically (visual fields and dark adapta-tion), and subsequently observe them in a "light laboratory": a standardizedenvironment in which a wide range of light intensities can be applied. The

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ultimate goal is to improve on the advice we give these patients with regard toseveral actvities, like driving, cycling in the dark, etc..

2. The most frequently established retinal disorder in our patients is TRD (seeChapter 2). We usually advise TRD patients with very constricted visual fields(less than 10º) not to cycle, and even use a white stick when walking. However,visual fields are measured under mesopic conditions, and therefore will not berepresentative of, for instance, a sunny day. To clarify this problem a Goldmannperimeter should be modified, to allow us to vary stimulus and backgroundconditions from scotopic to photopic intensities. The results of such visual fieldmeasurements with regard to daily activities will subsequently be evaluated. .

3. As described in Chapter 2, the prognosis of a condition may play a decisive rolein education and career choice. We therefore often get questions about thestability of a disorder. We have had several referrals of children with CSNB2, withsuspicion of deteriorating visual functions. To make certain the condition is asstationary as the name implies, we propose to investigate a large group of CSNB2patients that we have examined several years ago. We will compare their presentelectrophysiological and psychophysical functions with those measured earlier.A second problem is prescribing low vision aids in "stationary" conditions thatworsen, for instance early macular degeneration in achromatopsia and blue conemonochromatism. Patients may have an aversion of bright light because of theiroriginal condition, but need good illumination measures because of theirsecondary one. These patient groups are small, and therefore we plan to collectinformation from other rehabilitation centres to compare the results of differentways of rehabilitation, resulting in a protocol for prescribing low vision aids inthese cases.

4. At Bartiméus, we have a relatively high number of children with albinism. InChapter 6, we describe misrouting without albinism; the reverse of course alsoexists: albinism without misrouting. We plan to investigate this large patient groupwith regard to the presence or absence of misrouting, related to mutation analysis,foveal development and visual functions. We will also further investigate mis-routing in rare syndromes like Kartagener syndrome, and the foveal developmentin patients with achiasmia.

5. Because we regularly examine children with rare disorders, we will continue

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working together with centres for molecular genetics and biological researchcentres, which will hopefully result in discovering yet new genes and furtherclarification of retinal and postretinal processes.

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Chapter 9

Samenvatting



Samenvatting

Erfelijke netvliesaandoeningen en afwijkingen van de optische banen zijnbelangrijke oorzaken van slechtziendheid en blindheid bij kinderen. Elektrofysio-logisch onderzoek is vaak noodzakelijk om deze aandoeningen te diagnosticeren.Dit proefschrift laat het brede toepassingsgebied zien van kinder-electrofysiologieen geeft voorbeelden van de waarde van elektrofysiologie in het vaststellen vandiagnoses, het verklaren van pathofysiologische mechanismen, het voorspellen vanhet verloop van een aandoening en het onderbouwen van nieuwe hypothesen.

Hoofdstuk 2 beschrijft de resultaten van een retrospectieve studie van 340 elektro-fysiologische onderzoeken bij kinderen, verricht in een periode van drie jaar.Metingen werden gedaan zonder sedatie of anesthesie. Meerdere maatregelenwerden getroffen om de kans op een goed resultaat te vergroten. Bij ERG’sbestonden deze maatregelen uit het gebruik van DTL-elektroden, on-line selectievan responsies en het meten van een uitgebreid standaard ISCEV protocol. Deonderzochte kinderen hadden een gemiddelde leeftijd van 7 ± 5 jaar; slechts 4%van de onderzoeken mislukte wegens gebrek aan coöperatie. De meest gediagnos-ticeerde netvliesafwijkingen waren progressieve tapetoretinale dystrofieën,congenitale stationaire nachtblindheid en kegeldystrofie. Bij VEP metingen werdroutinematig misrouting bepaald, wat resulteerde in het ontdekken vanonverwachte misrouting bij zes kinderen. Bij 26 van de kinderen werd een eerdervastgestelde diagnose veranderd, waardoor bij 20 ook de prognose werd veran-derd. Deze veranderingen van diagnose hadden grote invloed op de revalidatie.

In hoofdstuk 3 wordt verslag gedaan van de oogheelkundige en elektrofysiologischebevindingen bij een 15-jarig meisje met een multi-systeem aandoening. Ze wasdoof, had suikerziekte en leed aan ernstige bloedarmoede waarvoor ze iedere tweemaanden een bloedtransfusie nodig had. Vanwege netvliesafwijkingen werd eenERG bepaald. Staafresponsies waren ernstig verlaagd, de kegelresponsiespraktisch afwezig, bewijzend voor kegel-staafdystrofie. De oogheelkundigebevindingen gecombineerd met de algemene afwijkingen leidden tot de diagnose"thiamine responsive megaloblastic anemia syndrome (TRMA: thiamine-afhankelijk megaloblastair anemie syndroom). Patiënten met TRMA hebben eenstoornis in het actieve thiamine transport, waardoor schade ontstaat aan meerdereorganen, waaronder de gehoorsbanen, de pancreas en het bloedvormend systeem.Retinale dystrofie was in een klein aantal gevallen beschreven. TRMA patiënten

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kunnen worden behandeld met farmacologische concentraties thiamine, omdathet passief opnemen van thiamine niet gestoord is. Behandeling van het meisje hadnormalisatie van de bloedcelwaarden en het hemoglobinegehalte tot gevolg,waardoor ze geen bloedtransfusies meer nodig had.

Hoofdstuk 4 beschrijft de oogheelkundige en elektrofysiologische bevindingen van24 patiënten met het Rubinstein-Taybi syndroom, een syndroom met meerderecongenitale afwijkingen en mentale retardatie. Onderzoek van de literatuur lieteen breed scala aan oogheelkundige afwijkingen zien, maar vooral van hetuitwendige oog, het voorsegment en de lens. Daarentegen had 78% van de 24 indeze studie onderzochte patiënten een niet goed functionerend netvlies; zo’n hogefrequentie was niet eerder beschreven. De afwijkende ERG’s vertoonden eenverminderde kegelrespons; de afwijkende VEP’s misten een striate component,wat wijst op een maculaire dysfunctie. Vier patiënten hadden normale ERG’s enVEP’s, wat duidt op fenotypische en mogelijk ook genotypische heterogeniteit.De retinale en elektrofysiologische afwijkingen kwamen met het stijgen van deleeftijd meer voor, wat een aanwijzing leek te zijn voor een progressieve retinaledystrofie.

In hoofdstuk 5 worden de klinische en oogheelkundige kenmerken beschreven vaneen meisje en haar jongere broertje met ectrodactylie, ectodermale dysplasie enmaculadystrofie (EEM syndroom). De kinderen waren de dertiende en veertiendepatiënt met EEM beschreven in 49 jaar, en de eersten bij wie de diagnose al werdvastgesteld op de zuigelingenleeftijd. Toen wij hen voor het eerst zagen, op deleeftijd van respectievelijk één jaar en van zeven maanden, hadden ze al uitgebreidenetvliesafwijkingen, een teken van een aangeboren netvliesdysplasie. Het uiterlijkvan het netvlies veranderde in tien jaar tijd nauwelijks en de ERG’s blevennormaal, wat past bij een stationaire aandoening en niet zozeer bij een maculairedystrofie. Het fundusbeeld vertoonde overeenkomsten met de netvliesafwijkingendie gezien worden bij ernstige pseudoxanthoma elasticum en zou daarom verbandkunnen houden met pathologie van de membraan van Bruch. EEM wordt veroorzaakt door mutaties in CDH3, wat codeert voor P-cadherine;mutaties in dit gen zijn ook verantwoordelijk voor hypotrichosis met juvenielemaculaire dystrofie (HJMD). De oogheelkundige kenmerken van de meeste EEMpatiënten, inclusief de onze, lijken verschillend van de afwijkingen welke in HJMDzijn beschreven, maar omdat sommige patiënten met een HJMD-achtigemaculadystrofie minimale hand- en tandafwijkingen hadden, zou het kunnen zijn

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dat EEM en HJMD fenotypische heterogeniteit van hetzelfde syndroomvertegenwoordigen.

In hoofdstuk 6 worden drie meisjes beschreven met misrouting en foveahypoplasie.Misrouting wordt beschouwd als hèt bewijs van albinisme en foveahypoplasie alseen belangrijk oogheelkundig kenmerk van deze aandoening. Echter, zowel depigmentatie als het retinale vaatpatroon van deze patiëntes pasten niet bijalbinisme. Eén van de meisjes had het Kartagenersyndroom, waarbij de locatie vande inwendige organen gespiegeld is (situs inversus). De resultaten duidden eropdat pigmentatie niet de enige bepalende factor is die misrouting en foveahypo-plasie tot gevolg heeft. De misrouting van deze patiëntes zou veroorzaakt kunnenzijn door afwijkingen in genen welke betrokken zijn bij de ontwikkeling van hetchiasma opticum. De relatie van misrouting met foveahypoplasie in verschillendeaandoeningen wijst op een retrograde beïnvloeding van de abnormale decussatieop de foveale ontwikkeling.

In hoofdstuk 7 worden de psychofysische, elektrofysiologische en genetischekenmerken beschreven van autosomaal recessief overervende congenitalestationaire nachtblindheid (arCSNB). De klinische kenmerken en de ISCEVstandaard ERG’s waren niet te onderscheiden van die van X-linked CSNB1("complete" CSNB). DNA analyse toonde in vijf van de zeven patiënten mutatiesaan in GRM6. GRM6 codeert de metabotrope glutamaatreceptor mGlur6, wat debelangrijkste receptor is van de ON bipolaire dendrieten. Menselijke staafsignalenmaken gebruik van twee wegen: de langzame "rod pathway", van staven naar staaf–ON-bipolaire cellen, en de snelle rod pathway, van de staven naar kegel-ON-bipolaire cellen, via staaf-kegel gap junctions. Deze twee rod pathways kunnenworden aangetoond door ERG metingen te doen met 15 Hz flikker stimulatie vanlage (scotopische) intensiteit. Bij afwezigheid van mGlur6 zouden we verwachtendat er geen signalen van beide ON pathways gemeten kunnen worden. Echterlangzame rod pathwaysignalen van de patiënten waren duidelijk meetbaar, maarmet een ongewoon fasegedrag met meerdere minima en faseomkeringen. Deresultaten wezen op het bestaan van alternatieve routes voor staafsignalen onderscotopische omstandigheden. De ongewone 15 Hz scotopische metingen werdenalleen gevonden in arCSNB patiënten met GRM6 mutaties; arCSNB patiëntenzonder mutaties en CSNB1 patiënten hadden geen meetbare langzame rodpathway signalen. 15 Hz scotopische flikker metingen zouden daarom gebruiktkunnen worden om deze aandoeningen van elkaar te onderscheiden.

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Elektrofysiologie en revalidatie; voorstellen voor verder onderzoek

In 2001 stelde de Wereld Gezondheidsorganisatie (WHO) de InternationaleClassificatie op van "Functioning, Disability and Health" (functioneren,beperking en gezondheid), afgekort ICF. Deze classificatie werd voor het eerstgemaakt in 1980 en toen de "International Classification of Impairments,Disabilities, and Handicaps" (ICIDH) genoemd. In tegenstelling tot ICIDH ishet taalgebruik van ICF neutraal ten opzichte van etiologie en legt meer de nadrukop functies dan op beperkingen of aandoeningen. ICF beschrijft het menselijkfunctioneren vanuit drie perspectieven:- functies en anatomische eigenschappen- activiteiten (het menselijk handelen) en participatie (deelname aan de

samenleving)- aanvullende informatie over ernst en omgevingsfactoren

Omdat onze elektrofysiologie-afdeling onderdeel is van een revalidatiecentrum,moet de afdeling niet alleen "functies en anatomische eigenschappen" vaststellen,maar belangrijk zijn voor de andere ICF gebieden: activiteiten, participatie enomgeving. In dit proefschrift zijn al enkele voorbeelden gegeven. In hoofdstuk 2worden de gevolgen van het bekend zijn van de prognose van een aandoeningbesproken, met betrekking tot scholing en beroepskeuze. Bij sommige Rubinstein-Taybi patiënten uit hoofdstuk 4 werd een visuele beperking vastgesteld welke hundagelijks leven zal beïnvloeden.

Gedurende de onderzoeken die in dit proefschrift beschreven staan stuitten weechter op meerdere nieuwe vragen die in de toekomst beantwoord moetenworden.

1. In hoofdstuk 7 onderzochten we patiënten met verschillende vormen vancongenitale stationaire nachtblindheid (CSNB). Het viel ons op dat het verbandtussen elektrofysiologische resultaten, onderzoek van visuele functies enproblemen met het zien in het donker bij meerdere soorten CSNB en tapetore-tinale dystrofie (TRD) niet eenduidig is. Patiënten met "complete " CSNB(CSNB1) hebben soms nauwelijks problemen met het zien in het donker, ondankseen volledig afwezige staaf-adaptatie in hun donkeradaptatiecurve. Patiënten met"incomplete"CSNB (CSNB2) hebben vaak meer problemen samenhangend meteen afwijkende kegelfunctie, zoals lichtschuwheid en gestoord kleurenzien, dan

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met afwijkende staaffunctie (reden waarom de term "incomplete" CSNB eenongelukkige is, omdat ouders de neiging hebben te denken dat incompleet mindererg is dan compleet). Echter bij beide aandoeningen kan het voorkomen datpatiënten met een ernstig gestoorde donkeradaptatie minder problemenondervinden dan patiënten met een betere donkeradaptatie. Bij TRD hebbenpatiënten vaak al vroeg in het verloop van de aandoening ernstige nachtblindheid,maar ook dit is wisselend. Een derde interessante aandoening in dit verband is eenvorm van recessief overervende CSNB met elektrofysiologisch afwezige (zowel aals b golf) staaffunctie maar volkomen normale kegelfunctie.We stellen voor deze patiëntengroepen elektrofysiologisch en psychofysisch teonderzoeken (gezichtsveldonderzoek en donkeradaptatie), en ze vervolgens teobserveren in een "lichtlab": een gestandaardiseerde omgeving waar een grotevariatie in lichtintensiteit kan worden gemaakt. Het uiteindelijke doel is derevalidatieadviezen te verbeteren die we nu aan deze patiënten geven, bijvoorbeeldmet betrekking tot autorijden en fietsen in het donker.

2. De meest voorkomende erfelijke netvliesafwijking in onze populatie is TRD (ziehoofdstuk 2). In het algemeen adviseren we TRD patiënten met ernstig beperktegezichtsvelden (minder dan 10º) niet te fietsen en zelfs een blindenstok tegebruiken bij het lopen. Gezichtsvelden worden echter gemeten ondermesopische omstandigheden, welke niet goed te vergelijken zijn met de omstan-digheden van bijvoorbeeld een zonnige dag. Wij stellen daarom voor eenGoldmann perimeter zodanig aan te passen, dat de lichtintensiteit van stimulus enachtergrondverlichting over een groot bereik gevarieerd kunnen worden.Vervolgens kunnen we dan de relatie bestuderen tussen de resultaten van dezegezichtsveldbepalingen en de dagelijkse activiteiten.

3. Zoals we in hoofdstuk 2 hebben beschreven, heeft de prognose van eenaandoening vaak een beslissende invloed op scholing en beroepskeuze. We krijgendaarom regelmatig vragen over de stabiliteit van een aandoening. Er zijn meerderekinderen met CSNB2 naar ons verwezen vanwege de verdenking op achteruitgangvan hun visuele functies. Om er zeker van te zijn dat de aandoening zo stationairis als de naam aangeeft, stellen we voor een aanzienlijke groep CSNB2 patiëntente onderzoeken die we een aantal jaren geleden ook hebben onderzocht. We zullenhun huidige elektrofysiologische en psychofysische functies met de vroegerevergelijken. Een tweede probleem is het voorschrijven van hulpmiddelen aan patiënten met

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verslechterende "stationaire" aandoeningen, bijvoorbeeld vroegtijdige maculade-generatie bij patiënten met achromatopsie of blauwe kegel monochromasie.Vanwege hun oorspronkelijke aandoening verdragen deze patiënten slecht licht,maar vanwege hun latere aandoening hebben ze juist veel licht nodig. Omdat hetom kleine patiëntengroepen gaat willen we inventariseren wat er op dit gebiedgebeurt, ook bij andere revalidatie-instellingen, om zo te komen tot een protocolvoor het voorschrijven van hulpmiddelen aan deze speciale patiëntengroepen.

4. We hebben een relatief grote populatie van kinderen met albinisme bijBartiméus. In hoofdstuk 6 beschrijven we het voorkomen van misrouting zonderalbinisme; het omgekeerde komt uiteraard ook voor: albinisme zonder misrouting.We zijn van plan deze relatief grote patiëntengroep te onderzoeken metbetrekking tot de aan- of afwezigheid van misrouting, gerelateerd aan DNAanalyse, foveaontwikkeling en visuele functies. We gaan ook door met hetonderzoeken van misrouting bij zeldzame syndromen zoals het Kartagenersyndroom, en de foveaontwikkeling van achiasmie patiënten.

5. Omdat we regelmatig kinderen zien met zeldzame aandoeningen, blijven weook in de toekomst samenwerken met centra voor moleculaire genetica enbiologisch onderzoek, wat hopelijk leidt tot de ontdekking van meer nogonbekende genen en verdere verheldering van retinale en postretinalemechanismen.

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Dankwoord

Graag wil ik allen bedanken die bijgedragen hebben aan het tot stand komen vandit proefschrift.

Allereerst de patiënten en hun families die steeds zo onbaatzuchtig bereid warenaan de onderzoeken mee te werken.

Mijn beide promotoren, Prof. Dr. F.M. Meire en Prof. Dr. J.S. Stilma, voor hunondersteuning. Beste Françoise, onze discussies waren altijd levendig envruchtbaar. Jouw grote kennis van de kinderoogheelkunde en genetica diende mijtot voorbeeld en inspiratie. Beste Jan, hartelijk dank dat je mij als "buitenpromo-vendus" hebt willen begeleiden. Jouw gestructureerde aanpak en kritische, snelle,maar vriendelijke commentaar hebben me zeer geholpen.

De leden van de beoordelingscommissie, Prof. Dr. D. van Norren. Prof. Dr.A.M.L. Kappers, Prof. Dr. J.J. de Laey, Prof. Dr. O. van Nieuwenhuizen en dr. D.Thompson voor de beoordeling van het manuscript.

Mijn co-promotor, dr. F.C.C. Riemslag. Beste Frans, jouw inbreng is van onschat-bare waarde geweest. Je leerde me alles over de elektrofysiologie en met je groteliefde voor dit vak stimuleer je iedereen die daarbij betrokken is. Voor ditproefschrift heb je eindeloos veel figuren bewerkt en berekeningen gemaakt, vaakcorrespondeerden we ver na middernacht nog per e-mail. Daarom wil ik ook jevrouw, Géke, bedanken voor alle tijd die ze jou heeft moeten inleveren.

Ir. F.F. Jorritsma en ir. F. P. Hoeben. Beste Franken, jullie waren altijd weer bereidextra werk te verrichten als ik daarom vroeg. De discussies in ons elektrofysio-logie-team zijn de basis geweest waaruit dit proefschrift tot stand is gekomen. Ikhoop dat we nog lang op deze inspirerende manier mogen samenwerken.

De afdeling oogheelkunde van Bartiméus waar veel mensen hebben bijgedragenaan dit werk: de orthoptistes Lia Brouwer, Marjoke Dekker, Heleen Veen, Anjavan Woerdekom en Inge Olsman die assisteerden bij de elektrofysiologie-procedures; het secretariaat onder leiding van Anneke van Staveren, dat steedsweer bereid was extra werk te verrichten, en mijn collega-oogartsen, in hetbijzonder José Schuil, die in tijden van grote drukte patiënten van me overnamen.

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Ook wil ik Piet Rison bedanken voor zijn inzet in het maken van foto’s en hetbrengen en halen van patiënten.

De bibliothecaresse, Anke de Jong en haar collega’s voor het onvermoeibaaraanleveren van grote hoeveelheden artikelen.

De Vereniging Bartiméus die de totstandkoming van dit proefschrift heeftgefinancierd; dank voor het in mij gestelde vertrouwen.

Mijn ouders, die altijd belangstelling hebben getoond, met me hebben meegeleefden me hebben ondersteund in goede en slechte tijden. Mijn zoons, Michiel enMenno, die nooit klaagden over de tijd die hun moeder niet besteedde aan hen,maar aan het schrijven van dit werk. Lieve zoons, met trots zie ik jullie opgroeienen zelfstandig worden.

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Curriculum vitae

Maria Michielde (Mies) van Genderen werd op 29 juni 1959 te Utrecht geboren engroeide vervolgens op in Doorn. In 1977 behaalde zij het diploma gymnasium-βaan het Revius Lyceum.

Van 1977-1979 studeerde zij Wis- en Natuurkunde aan de Universiteit vanUtrecht. In 1979 startte zij met de studie Geneeskunde, eveneens aan de Universi-teit van Utrecht; het doctoraal examen behaalde zij cum laude.

Na het artsexamen in 1986 volgde zij de opleiding tot oogarts in Leiden (opleidersprof. Dr. J.A. Oosterhuis en prof. C.C. Sterk), welke opleiding in juli 1991 werdvoltooid. Na kortstondig waarnemend oogarts te zijn geweest in Alphen a/d Rijn enAmersfoort, ging zij in februari 1992 werken bij Bartiméus. Vanaf 1999 heeft zijelektrofysiologie als speciaal interessegebied. In 2001 volgde zij de ISCEV Coursein Montréal, Canada.

De auteur heeft twee zoons: Michiel (1991) en Menno (1992).

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Documents

ELECTROPHYSIOLOGY IN VISUALLY IMPAIRED CHILDREN … · Introduction 1.1 Visual impairment in Dutch children The prevalence of childhood visual impairment in Northern en Western Europe,