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Clinical and Genetic Aspects of DFNA8/12, type III and Wolfram syndrome

R.F. Plantinga Print: Drukkerij Graficolor Nijmegen Lay-out: Diny Helsper

ISBN: 978-90-9021700-0 © R.F. Plantinga Titel proefschrift: Hereditary hearing impairment. Clinical and genetic aspects of DFNA8/12, Usher syndrome type III and Wolfram syndrome. Thesis Radboud University Nijmegen Medical Centre, Nijmegen. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechnically, by print or otherwise without written permission of the copyright owner. Hereditary Hearing Impairment

Clinical and Genetic Aspects of DFNA8/12, Usher syndrome type III and Wolfram syndrome

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het College van Decanen in het openbaar te verdedigen op vrijdag 1 juni 2007 om 10.30 uur precies

door

R.F. Plantinga geboren 16 oktober 1975 te Sneek Promotor: Prof. dr. C.W.R.J. Cremers

Copromotores: Dr. R.J.E. Pennings Dr. P.L.M. Huygen

Manuscriptcommissie: Prof.dr. B.C.J. Hamel Prof.dr. P. Van de Heyning (UZ Antwerpen) Prof.dr. J.R.M. Cruysberg

Publication of this thesis was financially supported by: Stichting Atze Spoor Fonds, ALK-Abelló, ALTHANA Pharma, Artu Biologicals, Atos Medical BV, Bayer BV, Beltone, Carl Zeiss BV, CenE Bankiers, Cochlear Benelux, Daleco Pharma BV, EmiD, Glaxo-Smith-Klein, HAL allergy BV, Olympus, Ooms allergie, Oticon, ReSound, Schering-Plough BV, Schoonenberg, UCB Pharma BV, Veenhuis Medical Audio BV, Veldhuizen Fokvarkens Table of contents

Chapter 1: General introduction 9

Chapter 2: DFNA8/12 (TECTA) 35 2.1 Van gen naar ziekte; DFNA8/12, een autosomaal dominant 37 overervend komvormig perceptief gehoorverlies. Cremers CWRJ, Plantinga RF, Kremer H Nederlands Tijdschrift voor Geneeskunde. 2007;151:531-534.

2.2: A novel TECTA mutation in a Dutch DFNA8/12 family confirms 47 genotype-phenotype correlation. Plantinga RF, de Brouwer APM, Huygen PLM, Kunst HPM, Kremer H, Cremers CWRJ Journal of the Association for Research in Otolaryngology 2006; 7: 173-181.

2.3: Audiological evaluation of affected members from a Dutch 65 DFNA8/12 (TECTA) family. Plantinga RF, Cremers CWRJ, Huygen PLM, Kunst HPM, Bosman AJ Journal of the Association for Research in Otolaryngology. 2007;8:1-7.

Chapter 3: Usher syndrome type III (USH3) 79 3.1: Serial and speech recognition findings in Finnish 81 Usher syndrome type III patients. Plantinga RF, Kleemola L, Huygen PLM, Joensuu T, Sankila E-M, Pennings RJE, Cremers CWRJ Audiology & Neuro-Otology 2005; 10: 79-89.

3.2: Visual impairment in Finnish Usher syndrome type III. 103 Plantinga RF, Pennings RJE, Huygen PLM, Sankila E-M, Tuppurainen K, Kleemola L, Cremers CWRJ, Deutman AF Acta Ophthalmologica Scandinavica 2006; 84: 36-41.

Chapter 4: Hearing impairment in genotyped Wolfram syndrome patients. 115 Plantinga RF, Pennings RJE, Huygen PLM, Bruno R, Eller P, Barrett TG, Vialettes B, Paquis-Fluklinger V, Lombardo F, Cremers CWRJ Submitted. Chapter 5: General discussion 129

Chapter 6: Summary / samenvatting 137 Acknowledgements 147 Curriculum Vitae 149 List of publications 151 Voor Mama

Chapter 1

General introduction

Chapter 1

Hereditary hearing impairment In recent history, remarkable progress has been made in our understanding of the basic principles of hereditary hearing impairment. The insight that heredity was a major cause of hearing impairment first appeared in the second edition of Adam Politzer’s Lehrbuch der Ohrenheilkunde, in 1887.1 Adam Politzer (1853-1920), an Austrian otologist, based his conclusions on the works of Wilde and Hartmann. Sir William Wilde (1815-1876), also an otologist, was medical commissioner during the Irish census in 1841 and the first to describe “direct” or dominant inheritance in hearing impairment.22 The German otologist Arthur Hartmann (1849-1931) was the first to describe the pattern of “indirect” or recessive inheritance in hearing impairment based on the, at that time relatively unappreciated, work of Gregor Mendel (1822-1884).3,4

In the nineteenth century, most research on hereditary hearing impairment concentrated on syndromes, such as Usher syndrome, with hearing impairment as one of the main features.5 With the introduction of the audiometer in the late 1930s, the non-syndromic types of hearing impairment slowly came into focus. The audiometer, which enabled physicians to assess the degree of hearing impairment, gradually became more widely available after the Second World War. In 1953 Watson and Crick opened up a new world with their discovery of the structure of DNA.6 However, it was not until 1992 that Léon et al. were the first to map the chromosomal locus for a gene responsible for nonsyndromic autosomal dominant hearing impairment.7

In neonates, genetic defects are responsible for at least 50% of the cases with hearing impairment. About 30% of those with hereditary hearing impairment have other associated anomalies, and about 70% have no other features - these are classified as syndromic and nonsyndromic, respectively. In nonsyndromic hereditary hearing impairment, the pattern of inheritance can be autosomal recessive (70- 80%), autosomal dominant (20-30%), X-linked (<1%), or mitochondrial (<1%). In general, nonsyndromic autosomal recessive hearing impairment is usually progressive and often more severe than nonsyndromic autosomal dominant hearing impairment.

Since the early 1990s, over 100 types of nonsyndromic sensorineural hearing impairment (SNHI) have been identified.8 The gene loci for nonsyndromic types of

11 General Introduction hearing impairment have been designated DFN () and are numbered in chronological order of discovery (e.g. DFNA1). Autosomal dominant types are referred to as DFNA, autosomal recessive types as DFNB, and X-linked types as DFN. Autosomal dominant types of hearing impairment especially can be characterised by audiometric configuration, age of onset (congenital, early, late onset), degree of progression, and severity of hearing impairment (according to the GENDEAF criteria: mild, 20-40 dB; moderate, 41-70 dB; severe, 71-95 dB; profound, >95dB).9

This PhD thesis focuses on DFNA8/12, Usher syndrome type III, and Wolfram syndrome; one type of nonsyndromic, autosomal dominant hearing impairment and two types of syndromic, autosomal recessive hearing impairment. This introduction will first concentrate on midfrequency hearing impairment, DFNA8/12, and subsequently, on Usher syndrome type III, and Wolfram syndrome.

Midfrequency hearing impairment Konigsmark and Gorlin (1976) were among the first to distinguish types of non- syndromic sensorineural hearing impairment by the shape of the audiogram.10 An example of a typical audiometric configuration is midfrequency hearing impairment, also described as “cookie-bite”, U-shaped, or trough-shaped configuration (Figure 1). This type of hearing impairment can be seen in families with DFNA8/12, DFNA13, DFNA21, DFNA31, DFNA44, and DFNA4911-20

dB -10 0

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Figure 1. Audiogram of individual with DFNA8/1221.

12 Chapter 1

Hearing loss primarily involving the mid-frequencies was first described in single families by Mårtensson (1960), and Williams and Roblee (1962), and later in additional families by Paparella et al. (1969) and Konigsmark et al. (1970).22-25 Paparella et al. also described temporal bone histopathology in a father and daughter with midfrequency hearing impairment.24 They observed a loss of ganglion cells, atrophy of the stria vascularis and degeneration of the organ of Corti.24 Sensorineural midfrequency hearing impairment is very rare.26

DFNA8/12 (TECTA) The first two reports on the genetics of non-syndromic, autosomal dominant mid- frequency hearing impairment describe an Austrian family (DFNA8) and a Belgian family (DFNA12).11-13 The proposed linkage to chromosome 15q in the Austrian family was later withdrawn and revised to chromosome 11q22-24.11,12 When the causative mutations for both families were shown to be located in the same gene (TECTA), the locus was designated as DFNA8/12.27 Subsequently, other mutations were found in families originating from Sweden, France, Spain, Japan and Turkey.28-32 Table 1 lists the reported families with their mutations, affected domains and phenotypes.

Table 1. DFNA8/12 TECTA mutations and their associated phenotype

Origin Mutation Domain Onset Phenotype Freq.

French29 C1619S ZA Variable Mild to moderate-severe, progressive High

Swedish28 C1057S ZA Postlingual Mild to severe, progressive High

Turkish32 C1509G ZA Prelingual Mild to moderate, progressive High

Spanish30 C1837G ZP Postlingual Mild to moderate, progressive Mid

Austrian11 Y1870C ZP Prelingual Moderate, stable Mid

Belgian12 L1820F, G1824D ZP Prelingual Moderate, stable Mid

Japanese31 R2021H ZP Prelingual Mild to moderate, stable Mid

The TECTA gene encodes the protein D-tectorin, an important noncollagenous component of the tectorial membrane in the cochlea. Alpha-Tectorin is expressed at high levels during tectorial membrane morphogenesis, between the 12th and 20th week of embryonic development, after which it decreases dramatically.12,32,33 The

13 General Introduction tectorial membrane consists of an extracellular matrix overlying the organ of Corti, and contacts the outer hair cells (Figure 2).

Figure 2. The organ of Corti.34

The D-tectorin protein is composed of three distinct modules: the entactin G1 domain, the zonadhesin (ZA) domain with von Willebrand factor type D repeats and the zona pellucida (ZP) domain.35 These domains are cross-linked to each other and E-tectorin by disulfide bridges to form the non-collagenous matrix of the tectorial membrane.35,36

Mutations affecting the ZP domain are associated with midfrequency hearing impairment, whereas mutations in the ZA domain are associated with hearing impairment primarily affecting the high frequencies (table 1).11,12,28-32 Additionally, mutations causing substitution of cysteine residues are associated with progressive hearing impairment (table 1).11,12,28-32

In 1999 Mustapha et al. analysed a large consanguineous family with autosomal recessive nonsyndromic, severe to profound hearing impairment and identified a new locus, designated DFNB21 on chromosome 11q23, the same region as TECTA.27 Sequence analysis of TECTA revealed a homozygous disease-causing mutation, thus demonstrating that mutations in TECTA can cause either dominant or recessive

14 Chapter 1 types of hearing impairment.27 The fact that heterozygous carriers of this mutation had normal hearing suggests that the mutations in DFNA8/12 exert a dominant- negative effect, while mutations in DFNB21 have a loss of function effect.27

TECTA mouse models Legan et al. studied mice deficient in Į-tectorin by a targeted disruption (½ENT) in Tecta that removes the entactin G1-like domain of Į-tectorin, as well as mice with an Y1870C mutation in the zona pellucida domain of Tecta.37,38 Mice homozygous for a targeted disruption (Tecta½ENT/½ENT) are functional nulls for Į-tectorin and have tectorial membranes that are detached from the cochlear sensory epithelium and lack all non-collagenous matrix, leading to hearing impairment, whereas heterozygous mice (Tecta½ENT/+) have normal tectorial membranes and normal hearing.37 Homozygosity for recessive, predicted null alleles of Į-tectorin in humans might exert a similar effect upon the tectorial membrane.39

In mice heterozygous for a Y1870C mutation (TectaY1870C/+) the matrix structure of the tectorial membrane is disrupted (Figure 3).38 Figures 3a-c show the light- microscopic analysis of the cochlea and organ of Corti in Tecta+/+, TectaY1870C/+, and TectaY1870C/Y1870C mice. In the TectaY1870C/+ mouse there is a considerable reduction in thickness of the limbal attachment zone (LZ) of the tectorial membrane, an absence of a marginal band (MB: a dense thickening running around the peripheral margin) and Hensen’s stripe (HS: a ridge that runs longitudinally along the underside of the tectorial membrane adjacent and parallel to the bundles of the inner hair cells), and detachment of Kimura’s membrane (arrowhead in Fig. 3b).38 In the TectaY1870C/Y1870C mouse, the tectorial membrane (arrow in Fig. 3c) is completely detached from the spiral limbus and the surface of the organ of Corti.38

The changes observed in the TectaY1870C/+ mouse do not seriously influence the tectorial membrane’s role in ensuring optimal cochlear feedback, however, neural thresholds were elevated and neural tuning curves were broadened.38 Therefore, the TectaY1870C/+ mouse may be a model for DFNA8/12(TECTA).

15 General Introduction

Figure 3a-c. Organ of Corti and tectorial membrane in mouse models.38 Light microscopic toluidine blue-stained, 1-Pm-thick sections of the cochlear duct. Fig 3a: limbal attachment zone (LZ) of the tectorial membrane, marginal band (MB), and Hensen’s stripe (HS). Fig 3b: detachment of Kimura’s membrane (arrowhead). Fig 3c: detached tectorial membrane (arrow). Bars: 50 Pm.

Chapter 2.1 presents a review on DFNA8/12 (in Dutch). Chapter 2.2 describes a new mutation in a Dutch DFNA8/12(TECTA) family, confirming the genotype-phenotype correlation. In chapter 2.3, an extensive and unique audiological evaluation of selected members from this family is presented to elucidate specific audiological features.

16 Chapter 1

Usher syndrome Usher syndrome, an eponym of the Scottish ophthalmologist Charles Howard Usher (1865-1942), is an autosomal recessive disorder that is characterised by the combination of SNHI and retinitis pigmentosa (RP). Usher syndrome was first described in 1858 by Albrecht von Graefe, a famous ophthalmologist, who reported on a patient and two of his sibs.5 Soon after, his student Richard Liebreich (1861), another German ophthalmologist, examined the deaf children of Berlin and observed a higher prevalence of the combination of RP and hearing impairment in the Jewish community.40 In those days, the autosomal recessive pattern of inheritance was not yet fully understood. Finally, in 1935 the disease was named after Charles Howard Usher, who dedicated his life to study visually impaired patients and their families and emphasized the hereditary nature of the disorder in his book entitled “on the inheritance of retinitis pigmentosa”.41

In 1977, Davenport and Omenn introduced a clinical classification of Usher syndrome and after several modifications it now comprises three different types (I-III) as is shown in table 2.42 Usher syndrome type I is characterised by congenital, profound SNHI, RP and vestibular areflexia. Usher syndrome type II shows moderate-to-severe congenital highfrequency SNHI, RP and intact vestibular responses. Usher syndrome type III generally shows progressive SNHI, RP and variable vestibular responses. RP leads to impaired dark adaptation and night blindness, progressive visual field constriction and reduction in visual acuity in all types of Usher syndrome and may result in blindness.

Table 2. Modified clinical classification of Davenport and Omenn.42

Type Hearing impairment Onset of RP Vestibular responses

I Congenital profound Onset in first decade Absent II Congenital sloping audiogram Onset in first or second decade Intact

III Progressive Variable Variable

In 2001 Otterstedde et al. proposed a new classification for Usher syndrome type I after the observation of intact vestibular function in profoundly hearing impaired patients.43 These clinically diagnosed Usher syndrome type I patients, however, were

17 General Introduction never genetically classified and may in fact be Usher syndrome type III patients with early onset of highly progressive sensorineural hearing impairment.43

Davenport et al. found that about 90% of reported cases had profound congenital deafness with onset of RP before puberty, whereas the rest had moderate to severe hearing impairment from birth and RP beginning after puberty.44 In 1989 Karjalainen et al. reported a remarkably high prevalence of progressive hearing impairment in Usher syndrome patients in Finland.45

Usher syndrome was estimated to occur in 3.0 per 100,000 in Scandinavia and 4.4 per 100,000 in the United States.46,47 The overall prevalence ranges from 3.5 to 6.2 per 100,000, making Usher syndrome the most common cause of deaf-blindness worldwide.47-51 European studies show a proportion of 25-44% of Usher syndrome type I and 56-75% of Usher syndrome type II48-51 A recent study by Kimberling et al. estimated that for the most common genetic subtypes USH1B and USH2A the population rate is approximately 1 in 70,000 and 1 in 17,000, respectively. The carrier frequency was estimated to be 1 in 132 for USH1B and 1 in 65 for USH2A.52

Since Kimberling et al. in 1990 mapped the first locus (USH2A) to chromosome 1q41, Usher syndrome has proved to be genetically heterogenous.8,53 Until now, 6 chromosomal loci have been identified for Usher type I (USH1B-G), two loci for Usher type II (USH2A, C) and only one locus for Usher type III (USH3A).8 At present, 8 genes (MYO7A, USH1C, CDH23, PCDH15, SANS, USH2A, VLGR1, and USH3A) have been identified (Table 3).8 Recently, the presumed USH1A locus on 14q32, as described by Kaplan et al.,54 was retracted because 7 out of the 9 reported families harboured mutations in the myosin VIIA gene, 1 was compatible with linkage to the USH1D and USH1E loci, and 1 excluded all USH1 loci (including the 14q32 region).55 Furthermore, the original USH2B family, mapped to chromosome 3p23-24 by Hmani et al., proved to be linked to USH2C.56,57 Table 3 provides an overview of the genetic subtypes of Usher syndrome.

The proteins encoded by the eight established Usher syndrome genes belong to different protein families. The USH1B protein is the molecular motor myosin VIIa.58 The USH1D and USH1F genes encode cell-to-cell adhesion proteins, cadherin 23 and protocadherin 15.62-64

18 Chapter 1

Table 3. Genetic subtypes of Usher syndrome.8

Type Genetic subtype Location Gene References I USH1B 11q13.5 MYO7A 58 USH1C 11p15.1 USH1C 59-61 USH1D 10q22.1 CDH23 62-64 USH1E 21q21 Unknown 65 USH1F 10q21-22 PCDH15 66,67 USH1G 17q24-25 SANS 68,69 II USH2A 1q41 USH2A 53,70,71 USH2C 5q14.3-q21.3 VLGR1 72,73 III USH3A 3q21-q25 USH3A 74,75

The USH1C and USH1G genes encode scaffold proteins harmonin and SANS.76,77 Usherin is a transmembrane protein that is involved in USH2A and the G-protein- coupled 7-transmembrane receptor (VLGR1) in USH2C.53,70-73 The USH3A gene codes for clarin-1, a member of the clarin family that has 4 transmembrane domains.78 Recent studies have shown that, although there are big differences in phenotype, the USH1 and USH2 proteins are integrated in an Usher protein interactome.79-82 The USH3A protein clarin-1 has, so far, not been proven to be part of the Usher protein interactome.80

Mutations in the different Usher genes can lead to a broad spectrum of phenotypes in the ear and eye, but there is increasing evidence for the existence of an integrated Usher protein network in both the inner ear and the retina.79,81,82 The Usher protein complex in inner ear and retina seems to have its major function in the neurosensory cells, respectively in the hair cells and photoreceptor cells.83 In the inner ear, hair bundles are located at the apical surface of both auditory and vestibular hair cells.83

The major sites of colocalization of Usher proteins in the inner ear are the stereocilia and the synaptic regions of hair cells.79,81,82,84,85 In addition, the spiral ganglion neurons harbour several of the Usher proteins such as usherin, protocadherin 15, and clarin-1.67,78,82 In the cuticular plate, harmonin and myosin VIIa are co-expressed.76,86-

19 General Introduction

88 Cadherin 23 is also found in Reissner’s membrane and SANS, protocadherin 15 and usherin in the supporting cells.67,81,89 In the retina, the Usher proteins co-localize in the outer plexiform layer of the photoreceptor, as well as in the ciliary region between the outer- and inner segments, and the calycal processes.77,79,80,82,86,90-93

Genotype-phenotype correlation In recent years, a lot of research has been performed on genotype-phenotype correlation in Usher syndrome patients, in order to provide clinicians with essential information to obtain a correct clinical diagnosis and to inform the patients and their relatives about the prognosis of their disease.

Usher syndrome type I patients have a congenital severe to profound SNHI, congenitally absent or severely diminished vestibular responses, and RP with subsequent impairment of vision, starting in the first decade of life. Later in childhood, patients develop decreased peripheral vision that progresses into tunnel vision over decades.94 Visual acuity also deteriorates with increasing age.94 Due to their vestibular areflexia, children with Usher syndrome type I have delayed developmental milestones and do not start walking until the age of 18-24 months. USH1B is the most common subtype with an estimated prevalence of 45-60% in Usher syndrome type I.52

Usher syndrome type II (USH2A) patients have congenital moderate to severe SNHI with a downsloping audiogram, normal vestibular responses and RP with onset in the first or second decade. However, intra and interfamilial variation in this phenotype can be observed. SNHI can be progressive by about 0.5 dB per year for all frequencies.95 Visual impairment increases similarly to USH1B with advancing age.94 Children with Usher syndrome type II usually have normal milestones. USH2A is the most common subtype (80%) of Usher syndrome type II. Figure 4 shows age-related typical audiograms (ARTA) for USH1B and USH2A.

In a retrospective study on visual impairment in patients with Usher syndrome types I and II, Sadeghi et al. found that progressive loss of visual acuity and visual field size attains a substantial degree between the second and third decade of life. They concluded that the rate of degeneration varies between individuals in both types of

20 Chapter 1

Usher syndrome. However, in this study around 70% retained good reading vision until the age of 60 years or more.98

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60 10 20 30 80 40 50 60 100 USH1B Threshold 120 0-60 .25 .5 1 2 4 8 kHz Frequency

Figure 4. ARTA of USH1B and USH2A (age in years in italics).96,97

The genetic subtypes USH1B and USH2A account for 75-80% of all Usher patients worldwide, whereas USH3A is rare (~2%).99,100 However, Usher syndrome type III is common in Scandinavian countries, especially in Finland.

Usher syndrome type III As mentioned before, Karjalainen et al. reported a remarkably high prevalence of progressive hearing impairment in Usher syndrome patients in Finland.45 A nationwide study conducted by Pakarinen et al. confirmed that Usher syndrome type III accounts for 40% of all Finnish Usher patients and therefore is the most common form of Usher syndrome in Finland.101 Formerly, Usher syndrome type III in Finland was called dystrophia retinae pigmentosa – dysacusis (DRD) syndrome or Nuutila disease.102 The high prevalence of this syndrome can be explained by taking notion of the Finnish Disease Heritage, a collection of nearly 40 rare hereditary diseases over-represented in Finland.102,103 The Finnish Disease Heritage is the result of the Finnish demographic history, which is characterised by the presence of

21 General Introduction national and regional isolates with rapid expansion.103 Specific mutations that probably have been imported by early immigrants have been enriched all over the country.104 The associated Finnish founder mutation identified in nearly all Usher syndrome type III cases is the Finmajor mutation in the USH3A gene on chromosome 3q21-q25 that changes codon for a tyrosine into a premature stop codon and leads to a truncated protein.75,78

The USH3A gene encodes the protein clarin-1, which is expressed in several tissues including the sensory epithelia of the auditory and visual system.75,78 In the inner ear, clarin-1 expression is found in the inner and outer hair cells of the organ of Corti and spiral ganglion cells that contain the primary neurons that innervate the cochlear sensory epithelia.78 Adato et al. suggested that clarin-1 has a role in the excitatory ribbon synapse junctions between hair cells and cochlear ganglion cells, as well as in analogous synapses within the retina.78,105

Pakarinen et al. studied 42 Usher syndrome type III patients and stated that hearing impairment in this type of Usher syndrome most often has a postlingual onset and progresses in 5-30 years to severe or profound levels.101 They also performed ophthalmologic examinations and reported these patients to have predominant hypermetropia with astigmatism, as opposed to hypermetropia without astigmatism in Usher syndrome type I patients and myopic refractive errors in Usher syndrome type II patients.106 Recently, Sadeghi et al. studied 28 patients with Usher syndrome type III and concluded that 35% of the patients had severe hearing impairment at an early age (4 to 6 years); progression began in the first decade and approximately 50% of the patients became profoundly deaf by the age of 40.107 Vestibular function was variable, ranging from normal to vestibular dysfunction.107 Patients with Usher syndrome type III can be misdiagnosed as having Usher syndrome type I or Usher syndrome type II.107,108

Chapters 3.1 and 3.2 of this thesis provide a detailed audiometric and ophthalmologic evaluation of Finnish Usher syndrome type III patients, resulting from close co- operation with our Finnish colleagues. Auditory and visual features were compared with data previously reported for Dutch USH1B and USH2A patients. The results of this study contribute to the understanding of the genotype-phenotype correlations in all types of Usher syndrome.

22 Chapter 1

Wolfram syndrome Wolfram syndrome is an autosomal recessive inherited deaf blindness syndrome that is characterized by Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness (DIDMOAD). Wolfram syndrome is rare, with an estimated prevalence of 1:770,000.109 The first report on the association of diabetes mellitus and optic atrophy in siblings was made by Wolfram and Wagner in 1938.110 In 1949 De Lawter added the feature .111 Tunbridge, in his 1956 study on the syndromic association, reported on Wagner’s unpublished observation that 3 out of 4 patients had subnormal hearing.110,112 In 1977, Cremers et al. named the association Wolfram syndrome in their systematic literature review of 88 cases.113

Insulin-dependent diabetes mellitus and bilateral optic atrophy are the minimal features necessary to establish the diagnosis of Wolfram syndrome. Typically, diabetes mellitus is detected first at a mean age of 6 years, followed by optic atrophy at a mean age of 11 years. Hypothalamic diabetes insipidus, which occurs in 73% of the patients, and SNHI (62%) usually present in the second decade of life.109 In addition to these four classical features, patients can develop renal tract complications (58%) in the third decade, neurological complications such as cerebellar ataxia or myoclonus (62%) in the fourth decade, behavioural and psychiatric illness (60%), gastro-intestinal dysmotility (24%) and primary gonad atrophy.109,114 Swift et al. reported that relatives of Wolfram syndrome patients who are a heterozygous carrier of a WFS1 mutation, are 26-fold more likely to require psychiatric hospitalization than non-carriers.115 Later, the same authors estimated the relative risk for admission for psychiatric illness to be 7.1.116 Other reported features are upper gastrointestinal ulceration and bleeding, malformations and anterior pituitary dysfunction.117,118 In general, Wolfram syndrome represents a progressive neurodegenerative disorder that affects central, peripheral and neuroendocrine components of the nervous system resulting in death around the age of 40.119

In 1998, two research groups reported the WFS1 gene, located on chromosome 4p16.1, to be involved in Wolfram syndrome.120,121 WFS1 encodes wolframin, a transmembrane protein that consists of 890 amino acids.121 Wolframin is expressed in the endoplasmatic reticulum of a number of cell types in heart, , brain and .121,122 Immunostaining in developing murine inner ear showed that wolframin localizes to the canalicular reticulum, a specialized form of

23 General Introduction endoplasmatic reticulum.123 The canalicular reticulum has been identified in various types of inner ear cells, including vestibular and cochlear hair cells, spiral ganglion cells, Deiter’s cells and cells in the stria vascularis and is suggested to be involved in intercellular ion transport, an important process in the physiology of the inner ear.119,123

The discovery of the WFS1 gene weakened the earlier hypothesis that deletions and pathogenic point mutations in mitochondrial DNA are responsible for Wolframe syndrome.124,126 In 2000 Barrett et al. investigated a cohort of 50 patients, and found no abnormal mitochondrial (mt) function or mtDNA mutations and concluded that there is no evidence supporting a role for mtDNA in Wolfram syndrome.127 This was confirmed in a study by Domenech et al. who identified WFS1 mutations in six Wolfram syndrome families without evidence of mitochondrial mutations.128

Genotype-phenotype correlation Mutations in WFS1 can also result in an autosomal dominant form of non-syndromic SNHI classified as DFNA6/14/38.129,130 In contrast to Wolfram syndrome, DFNA6/14/38 is characterized by low-frequency SNHI.131 Hearing impairment in DFNA6/14/38 can be either progressive or non-progressive.131 Mutation analysis of WFS1 in families with DFNA6/14/38 revealed that the identified pathogenic mutations in this disorder all are small non-inactivating (missense) mutations, whereas most of the pathogenic mutations in Wolfram syndrome patients are inactivating mutations.132 The on-line WFS1 gene mutation and polymorphism database lists all documented mutations for DFNA6/14/38 and Wolfram syndrome.133

The majority of mutations underlying Wolfram syndrome are nonsense, frameshift, or significant insertions or deletions expected to produce a functionally null protein. However, a significant proportion of the mutations in Wolfram syndrome are missense or small in-frame insertions or deletions expected to produce a functional protein. This lead McHugh and Friedman to hypothesize that the phenotypic spectrum of Wolfram syndrome is also influenced by modifier genes.119

El-Shanti et al. found a second locus for Wolfram syndrome (WFS2) in three Jordanian families, located on chromosome 4q22-24.117 All affected family members

24 Chapter 1 were diagnosed with diabetes mellitus and optic atrophy, but remarkably, none of theses patients had diabetes insipidus.117

Hearing impairment in Wolfram syndrome In 2004, Pennings et al. reported on 11 Dutch Wolfram syndrome patients with confirmed mutations in WFS1.134 They concluded that progression in hearing impairment mainly occurred in the mid- and high-frequency range.134 In addition, they observed that the hearing impairment in 5 female patients with Wolfram syndrome was substantially worse when compared to the hearing impairment in 4 male patients.134 They hypothesised that this gender-related difference in hearing impairment might be explained by the involvement of estrogen.134 Figure 5 shows the binaural mean audiogram of five female and four male patients.

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Figure 5. Gender-related hearing impairment in Wolfram patients. Patients are represented with squares (male) and circles (females).134

To further outline the feature of sensorineural hearing impairment in Wolfram syndrome in terms of presence and degree of progression and degree of variability and to evaluate whether the degree of hearing impairment in Wolfram syndrome patients is gender-related an international multicentre study was conducted.

25 General Introduction

Chapter 4 of this thesis provides a comprehensive evaluation of hearing impairment in this multicentre study of genotyped Wolfram syndrome patients.

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27 General Introduction

31. Iwasaki S, Harada D, Usami S, Nagura M, Takeshita T, Hoshino T. Association of clinical features with mutation of TECTA in a family with autosomal dominant hearing loss. Arch Otolaryngol Head Neck Surg 2002;128:913-917. 32. Pfister MHF, Thiele H, Van Camp G, Fransen E, Apaydin F, Aydin O, Leistenschneider P, Devoto M, Zenner HP, Blin N, Nurnberg P, Ozkarakas H, Kupka S. A genotype-phenotype correlation with gender-effect for hearing impairment caused by TECTA mutations. Cell Physiol Biochem 2004;14:369-376. 33. Govaerts PJ, De Ceulaer G, Daemers K, Verhoeven K, Van Camp G, Schatteman I, Verstreken M, Willems PJ, Somers T, Offeciers FE. A new autosomal-dominant locus (DFNA12) is responsible for a nonsyndromic, midfrequency, prelingual and nonprogressive sensorineural hearing loss. Am J Otol 1998;19:718-723. 34. 'Ear, Human'. Encyclopaedia Britannica . 2004. Ref Type: Electronic Citation 35. Legan PK, Rau A, Keen JN, Richardson GP. The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system. J Biol Chem 1997;272:8791-8801. 36. Killick R, Legan PK, Malenczak C, Richardson GP. Molecular cloning of chick beta-tectorin, an extracellular matrix molecule of the inner ear. J Cell Biol 1995;129:535-547. 37. Legan PK, Lukashkina VA, Goodyear RJ, Kossi M, Russell IJ, Richardson GP. A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 2000;28:273-285. 38. Legan PK, Lukashkina VA, Goodyear RJ, Lukashkin AN, Verhoeven K, Van Camp G, Russell IJ, Richardson GP. A deafness mutation isolates a second role for the tectorial membrane in hearing. Nat Neurosci 2005;8:1035-1042. 39. Naz S, Alasti F, Mowjoodi A, Riazuddin S, Sanati MH, Friedman TB, Griffith AJ, Wilcox ER, Riazuddin S. Distinctive audiometric profile associated with DFNB21 alleles of TECTA. J Med Genet 2003;40:360-363. 40. Liebreich R. Abkunft aus Ehen unter Blutsverwandten als Grund von Retinitis pigmentosa. Dtsch Arch Klin Med 1861;53-55. 41. Usher CH. On the inheritance of retinitis pigmentosa, with notes of cases. R Lond Ophthalmol Hosp Rep 1916;19:130-236. 42. Davenport SLH, Omenn GS. The heterogeneity of Usher syndrome. In: Littlefield JW, Ebbing FJG, Henderson JW, eds. Fifth International Conference on Birth Defects. Amsterdam, Excerpta Medica. 1977: 87-88. 43. Otterstedde CR, Spandau U, Blankenagel A, Kimberling WJ, Reisser C. A new clinical classification for Usher's syndrome based on a new subtype of Usher's syndrome type I. Laryngoscope 2001;111:84-86. 44. Davenport SL, O'Nuallain S, Omenn GS, Wilkus RJ. Usher syndrome in four hard-of-hearing siblings. Pediatrics 1978;62:578-583. 45. Karjalainen S, Pakarinen L, Terasvirta M, Kaariainen H, Vartiainen E. Progressive hearing loss in Usher's syndrome. Ann Otol Rhinol Laryngol 1989;98:863-866. 46. Hallgren B. Retinitis pigmentosa combined with congenital deafness; with vestibulo-cerebellar ataxia and mental abnormality in a proportion of cases: A clinical and genetico-statistical study. Acta Psychiatr Scand 1959;34(Suppl 138):1-101.

28 Chapter 1

47. Boughman JA, Vernon M, Shaver KA. Usher syndrome: definition and estimate of prevalence from two high-risk populations. J Chronic Dis 1983;36:595-603. 48. Grøndahl J. Estimation of prognosis and prevalence of retinitis pigmentosa and Usher syndrome in Norway. Clin Genet 1987;31:255-264. 49. Hope CI, Bundey S, Proops D, Fielder AR. Usher syndrome in the city of Birmingham--prevalence and clinical classification. Br J Ophthalmol 1997;81:46-53. 50. Rosenberg T, Haim M, Hauch AM, Parving A. The prevalence of Usher syndrome and other retinal dystrophy-hearing impairment associations. Clin Genet 1997;51:314-321. 51. Spandau UH, Rohrschneider K. Prevalence and geographical distribution of Usher syndrome in Germany. Graefes Arch Clin Exp Ophthalmol 2002;240:495-498. 52. Kimberling WJ. Estimation of the frequency of occult mutations for an autosomal recessive disease in the presence of genetic heterogeneity: application to genetic hearing loss disorders. Hum Mutat 2005;26:462-470. 53. Kimberling WJ, Weston MD, Moller C, Davenport SL, Shugart YY, Priluck IA, Martini A, Milani M, Smith RJH. Localization of Usher syndrome type II to chromosome 1q. Genomics 1990;7:245- 249. 54. Kaplan J, Gerber S, Bonneau D, Rozet JM, Delrieu O, Briard ML, Dollfus H, Ghazi I, Dufier JL, Frezal J, . A gene for Usher syndrome type I (USH1A) maps to chromosome 14q. Genomics 1992;14:979-987. 55. Gerber S, Bonneau D, Gilbert B, Munnich A, Dufier JL, Rozet JM, Kaplan J. USH1A: Chronicle of a Slow Death. Am J Hum Genet 2006;78:357-359. 56. Gaehel, I. members of the Eurohear consortium. 2006. Ref Type: Personal Communication 57. Hmani M, Ghorbel A, Boulila-Elgaied A, Ben ZZ, Kammoun W, Drira M, Chaabouni M, Petit C, Ayadi H. A novel locus for Usher syndrome type II, USH2B, maps to chromosome 3 at p23-24.2. Eur J Hum Genet 1999;7:363-367. 58. Weil D, Blanchard S, Kaplan J, Guilford P, Gibson F, Walsh J, Mburu P, Varela A, Levilliers J, Weston MD, . Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature 1995;374:60-61. 59. Smith RJH, Lee EC, Kimberling WJ, Daiger SP, Pelias MZ, Keats BJ, Jay M, Bird A, Reardon W, Guest M, . Localization of two genes for Usher syndrome type I to chromosome 11. Genomics 1992;14:995-1002. 60. Verpy E, Leibovici M, Zwaenepoel I, Liu XZ, Gal A, Salem N, Mansour A, Blanchard S, Kobayashi I, Keats BJ, Slim R, Petit C. A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C. Nat Genet 2000;26:51-55. 61. Bitner-Glindzicz M, Lindley KJ, Rutland P, Blaydon D, Smith VV, Milla PJ, Hussain K, Furth-Lavi J, Cosgrove KE, Shepherd RM, Barnes PD, O'Brien RE, Farndon PA, Sowden J, Liu XZ, Scanlan MJ, Malcolm S, Dunne MJ, Aynsley-Green A, Glaser B. A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene. Nat Genet 2000;26:56-60. 62. Wayne S, Der Kaloustian VM, Schloss M, Polomeno R, Scott DA, Hejtmancik JF, Sheffield VC, Smith RJH. Localization of the Usher syndrome type ID gene (Ush1D) to chromosome 10. Hum Mol Genet 1996;5:1689-1692.

29 General Introduction

63. Bork JM, Peters LM, Riazuddin S, Bernstein SL, Ahmed ZM, Ness SL, Polomeno R, Ramesh A, Schloss M, Srisailpathy CR, Wayne S, Bellman S, Desmukh D, Ahmed Z, Khan SN, Kaloustian VM, Li XC, Lalwani A, Riazuddin S, Bitner-Glindzicz M, Nance WE, Liu XZ, Wistow G, Smith RJH, Griffith AJ, Wilcox ER, Friedman TB, Morell RJ. Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin- like gene CDH23. Am J Hum Genet 2001;68:26-37. 64. Bolz H, von Brederlow B, Ramirez A, Bryda EC, Kutsche K, Nothwang HG, Seeliger M, del C- Salcedo Cabrera, Vila MC, Molina OP, Gal A, Kubisch C. Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D. Nat Genet 2001;27:108- 112. 65. Chaib H, Kaplan J, Gerber S, Vincent C, Ayadi H, Slim R, Munnich A, Weissenbach J, Petit C. A newly identified locus for Usher syndrome type I, USH1E, maps to chromosome 21q21. Hum Mol Genet 1997;6:27-31. 66. Ahmed ZM, Riazuddin S, Bernstein SL, Ahmed Z, Khan S, Griffith AJ, Morell RJ, Friedman TB, Riazuddin S, Wilcox ER. Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F. Am J Hum Genet 2001;69:25-34. 67. Alagramam KN, Yuan H, Kuehn MH, Murcia CL, Wayne S, Srisailpathy CR, Lowry RB, Knaus R, Van Laer L, Bernier FP, Schwartz S, Lee C, Morton CC, Mullins RF, Ramesh A, Van Camp G, Hageman GS, Woychik RP, Smith RJH. Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F. Hum Mol Genet 2001;10:1709-1718. 68. Mustapha M, Chouery E, Torchard-Pagnez D, Nouaille S, Khrais A, Sayegh FN, Megarbane A, Loiselet J, Lathrop M, Petit C, Weil D. A novel locus for Usher syndrome type I, USH1G, maps to chromosome 17q24-25. Hum Genet 2002;110:348-350. 69. Weil D, El-Amraoui A, Masmoudi S, Mustapha M, Kikkawa Y, Laine S, Delmaghani S, Adato A, Nadifi S, Zina ZB, Hamel C, Gal A, Ayadi H, Yonekawa H, Petit C. Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin. Hum Mol Genet 2003;12:463-471. 70. Eudy JD, Weston MD, Yao S, Hoover DM, Rehm HL, Ma-Edmonds M, Yan D, Ahmad I, Cheng JJ, Ayuso C, Cremers CWRJ, Davenport S, Moller C, Talmadge CB, Beisel KW, Tamayo M, Morton CC, Swaroop A, Kimberling WJ, Sumegi J. Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa. Science 1998;280:1753-1757. 71. van Wijk E, Pennings RJE, Te Brinke H, Claassen A, Yntema HG, Hoefsloot LH, Cremers FP, Cremers CWRJ, Kremer H. Identification of 51 novel exons of the Usher syndrome type 2A (USH2A) gene that encode multiple conserved functional domains and that are mutated in patients with Usher syndrome type II. Am J Hum Genet 2004;74:738-744. 72. Pieke-Dahl S, Moller CG, Kelley PM, Astuto LM, Cremers CWRJ, Gorin MB, Kimberling WJ. Genetic heterogeneity of Usher syndrome type II: localisation to chromosome 5q. J Med Genet 2000;37:256-262. 73. Weston MD, Luijendijk MW, Humphrey KD, Moller C, Kimberling WJ. Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of Usher syndrome type II. Am J Hum Genet 2004;74:357-366.

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74. Sankila EM, Pakarinen L, Kaariainen H, Aittomaki K, Karjalainen S, Sistonen P, de la Chapelle A. Assignment of an Usher syndrome type III (USH3) gene to chromosome 3q. Hum Mol Genet 1995;4:93-98. 75. Joensuu T, Hamalainen R, Yuan B, Johnson C, Tegelberg S, Gasparini P, Zelante L, Pirvola U, Pakarinen L, Lehesjoki AE, de la Chapelle A, Sankila EM. Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3. Am J Hum Genet 2001;69:673-684. 76. Boeda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, Blanchard S, Perfettini I, Fath KR, Shorte S, Reiners J, Houdusse A, Legrain P, Wolfrum U, Richardson G, Petit C. Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. EMBO J 2002;21:6689-6699. 77. Reiners J, Marker T, Jurgens K, Reidel B, Wolfrum U. Photoreceptor expression of the Usher syndrome type 1 protein protocadherin 15 (USH1F) and its interaction with the scaffold protein harmonin (USH1C). Mol Vis 2005;11:347-355. 78. Adato A, Vreugde S, Joensuu T, Avidan N, Hamalainen R, Belenkiy O, Olender T, Bonne-Tamir B, Ben-Asher E, Espinos C, Millan JM, Lehesjoki AE, Flannery JG, Avraham KB, Pietrokovski S, Sankila EM, Beckmann JS, Lancet D. USH3A transcripts encode clarin-1, a four-transmembrane- domain protein with a possible role in sensory synapses. Eur J Hum Genet 2002;10:339-350. 79. Reiners J, van Wijk E, Marker T, Zimmermann U, Jurgens K, Te Brinke H, Overlack N, Roepman R, Knipper M, Kremer H, Wolfrum U. Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2. Hum Mol Genet 2005;14:3933-3943. 80. Reiners J, Nagel-Wolfrum K, Jurgens K, Marker T, Wolfrum U. Molecular basis of human Usher syndrome: Deciphering the meshes of the Usher protein network provides insights into the pathomechanisms of the Usher disease. Exp Eye Res 2006. 81. Adato A, Michel V, Kikkawa Y, Reiners J, Alagramam KN, Weil D, Yonekawa H, Wolfrum U, El- Amraoui A, Petit C. Interactions in the network of Usher syndrome type 1 proteins. Hum Mol Genet 2005;14:347-356. 82. van Wijk E, van der Zwaag B, Peters T, Zimmermann U, Te Brinke H, Kersten FF, Marker T, Aller E, Hoefsloot LH, Cremers CWRJ, Cremers FP, Wolfrum U, Knipper M, Roepman R, Kremer H. The DFNB31 gene product whirlin connects to the Usher protein network in the cochlea and retina by direct association with USH2A and VLGR1. Hum Mol Genet 2006;15:751-765. 83. Kremer H, van Wijk E, Marker T, Wolfrum U, Roepman R. Usher syndrome: Molecular links of pathogenesis, proteins and pathways. Hum Mol Genet 2006;15 Spec No 2:R262-R270. 84. Siemens J, Kazmierczak P, Reynolds A, Sticker M, Littlewood-Evans A, Muller U. The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions. Proc Natl Acad Sci U S A 2002;99:14946-14951. 85. Senften M, Schwander M, Kazmierczak P, Lillo C, Shin JB, Hasson T, Geleoc GS, Gillespie PG, Williams D, Holt JR, Muller U. Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells. J Neurosci 2006;26:2060-2071. 86. Wolfrum U, Liu X, Schmitt A, Udovichenko IP, Williams DS. Myosin VIIa as a common component of cilia and microvilli. Cell Motil Cytoskeleton 1998;40:261-271. 87. Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker MS. Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B. Proc Natl Acad Sci U S A 1995;92:9815-9819.

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88. Self T, Mahony M, Fleming J, Walsh J, Brown SD, Steel KP. Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells. Development 1998;125:557-566. 89. Adato A, Lefevre G, Delprat B, Michel V, Michalski N, Chardenoux S, Weil D, El-Amraoui A, Petit C. Usherin, the defective protein in Usher syndrome type IIA, is likely to be a component of interstereocilia ankle links in the inner ear sensory cells. Hum Mol Genet 2005;14:3921-3932. 90. Reiners J, Reidel B, El-Amraoui A, Boeda B, Huber I, Petit C, Wolfrum U. Differential distribution of harmonin isoforms and their possible role in Usher-1 protein complexes in mammalian photoreceptor cells. Invest Ophthalmol Vis Sci 2003;44:5006-5015. 91. Goodyear RJ, Richardson GP. Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity. J Neurobiol 2002;53:212- 227. 92. Goodyear RJ, Richardson GP. A novel antigen sensitive to calcium chelation that is associated with the tip links and kinocilial links of sensory hair bundles. J Neurosci 2003;23:4878-4887. 93. Liu X, Vansant G, Udovichenko IP, Wolfrum U, Williams DS. Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells. Cell Motil Cytoskeleton 1997;37:240-252. 94. Pennings RJE, Huygen PLM, Orten DJ, Wagenaar M, Van Aarum A, Kremer H, Kimberling WJ, Cremers CWRJ, Deutman AF. Evaluation of visual impairment in Usher syndrome 1b and Usher syndrome 2a. Acta Ophthalmol Scand 2004;82:131-139. 95. Pennings RJE, Huygen PLM, Weston MD, Van Aarum A, Wagenaar M, Kimberling WJ, Cremers CWRJ. Pure tone hearing thresholds and speech recognition scores in Dutch patients carrying mutations in the USH2A gene. Otol Neurotol 2003;24:58-63. 96. Wagenaar M, Van Aarum A, Huygen PLM, Pieke-Dahl S, Kimberling WJ, Cremers CWRJ. Hearing impairment related to age in Usher syndrome types 1B and 2A. Arch Otolaryngol Head Neck Surg 1999;125:441-445. 97. Plantinga RF, Kleemola L, Huygen PLM, Joensuu T, Sankila EM, Pennings RJE, Cremers CWRJ. Serial audiometry and speech recognition findings in Finnish Usher syndrome type III patients. Audiol Neurootol 2005;10:79-89. 98. Sadeghi M, Eriksson K, Kimberling WJ, Sjöström A, Moller CG. Long-Term Visual Prognosis in Usher Syndrome Type I and II. 1-21. 2005. Ref Type: Thesis/Dissertation 99. Pennings RJE, Wagenaar M, Van Aarum A, Huygen PLM, Kimberling WJ, Cremers CWRJ. Hearing impairment in Usher's syndrome. Adv Otorhinolaryngol 2002;61:184-191. 100. Aller E, Jaijo T, Oltra S, Alio J, Galan F, Najera C, Beneyto M, Millan JM. Mutation screening of USH3 gene (clarin-1) in Spanish patients with Usher syndrome: low prevalence and phenotypic variability. Clin Genet 2004;66:525-529. 101. Pakarinen L, Karjalainen S, Simola KO, Laippala P, Kaitalo H. Usher's syndrome type 3 in Finland. Laryngoscope 1995;105:613-617. 102. Norio R. The Finnish Disease Heritage III: the individual diseases. Hum Genet 2003;112:470-526. 103. Norio R, Nevanlinna HR, Perheentupa J. Hereditary diseases in Finland; rare flora in rare soul. Ann Clin Res 1973;5:109-141. 104. Peltonen L, Jalanko A, Varilo T. Molecular genetics of the Finnish disease heritage. Hum Mol Genet 1999;8:1913-1923.

32 Chapter 1

105. Wagner HJ. Presynaptic bodies ("ribbons"): from ultrastructural observations to molecular perspectives. Cell Tissue Res 1997;287:435-446. 106. Pakarinen L, Tuppurainen K, Laippala P, Mantyjarvi M, Puhakka H. The ophthalmological course of Usher syndrome type III. Int Ophthalmol 1995;19:307-311. 107. Sadeghi M, Cohn ES, Kimberling WJ, Tranebjaerg L, Moller C. Audiological and vestibular features in affected subjects with USH3: a genotype/phenotype correlation. Int J Audiol 2005;44:307-316. 108. Pennings RJE, Fields RR, Huygen PLM, Deutman AF, Kimberling WJ, Cremers CWRJ. Usher syndrome type III can mimic other types of Usher syndrome. Ann Otol Rhinol Laryngol 2003;112:525-530. 109. Barrett TG, Bundey SE, Macleod AF. Neurodegeneration and diabetes: UK nationwide study of Wolfram (DIDMOAD) syndrome. Lancet 1995;346:1458-1463. 110. Wolfram DJ, Wagner HP. Diabetes mellitus and simple optic atrophy among siblings: report of four cases. Proc Mayo Clin 1938;13:242-250. 111. De Lawter DE. Coexistence of diabetes insipidus and diabetes mellitus. Med Ann Dist Columbia 1949;18:393-400. 112. Tunbridge RE, Paley RG. Primary Optic Atrophy in Diabetes Mellitus. Diabetes 1956;5:295-296. 113. Cremers CWRJ, Wijdeveld PGAB, Pinckers AJLG. Juvenile diabetes mellitus, optic atrophy, hearing loss, diabetes insipidus, atonia of the urinary tract and bladder, and other abnormalities (Wolfram syndrome). A review of 88 cases from the literature with personal observations on 3 new patients. Acta Paediatr Scand Suppl 1977;1-16. 114. Swift RG, Sadler DB, Swift M. Psychiatric findings in Wolfram syndrome homozygotes. Lancet 1990;336:667-669. 115. Swift RG, Polymeropoulos MH, Torres R, Swift M. Predisposition of Wolfram syndrome heterozygotes to psychiatric illness. Mol Psychiatry 1998;3:86-91. 116. Swift M, Swift RG. Wolframin mutations and hospitalization for psychiatric illness. Mol Psychiatry 2005;10:799-803. 117. El-Shanti H, Lidral AC, Jarrah N, Druhan L, Ajlouni K. Homozygosity mapping identifies an additional locus for Wolfram syndrome on chromosome 4q. Am J Hum Genet 2000;66:1229- 1236. 118. Medlej R, Wasson J, Baz P, Azar S, Salti I, Loiselet J, Permutt A, Halaby G. Diabetes mellitus and optic atrophy: a study of Wolfram syndrome in the Lebanese population. J Clin Endocrinol Metab 2004;89:1656-1661. 119. McHugh RK, Friedman RA. Genetics of hearing loss: Allelism and modifier genes produce a phenotypic continuum. Anat Rec A Discov Mol Cell Evol Biol 2006;288:370-381. 120. Inoue H, Tanizawa Y, Wasson J, Behn P, Kalidas K, Bernal-Mizrachi E, Mueckler M, Marshall H, Donis-Keller H, Crock P, Rogers D, Mikuni M, Kumashiro H, Higashi K, Sobue G, Oka Y, Permutt MA. A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome). Nat Genet 1998;20:143-148. 121. Strom TM, Hortnagel K, Hofmann S, Gekeler F, Scharfe C, Rabl W, Gerbitz KD, Meitinger T. Diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD) caused by mutations in a novel gene (wolframin) coding for a predicted transmembrane protein. Hum Mol Genet 1998;7:2021-2028.

33 General Introduction

122. Takeda K, Inoue H, Tanizawa Y, Matsuzaki Y, Oba J, Watanabe Y, Shinoda K, Oka Y. WFS1 (Wolfram syndrome 1) gene product: predominant subcellular localization to endoplasmic reticulum in cultured cells and neuronal expression in rat brain. Hum Mol Genet 2001;10:477- 484. 123. Cryns K, Thys S, Van Laer L, Oka Y, Pfister M, Van Nassua L, Smith RJH, Timmermans JP, Van Camp G. The WFS1 gene, responsible for low frequency sensorineural hearing loss and Wolfram syndrome, is expressed in a variety of inner ear cells. Histochem Cell Biol 2003;119:247-256. 124. Rotig A, Cormier V, Chatelain P, Francois R, Saudubray JM, Rustin P, Munnich A. Deletion of mitochondrial DNA in a case of early-onset diabetes mellitus, optic atrophy and deafness (DIDMOAD, Wolfram syndrome). J Inherit Metab Dis 1993;16:527-530. 125. Bu X, Rotter JI. Wolfram syndrome: a mitochondrial-mediated disorder? Lancet 1993;342:598- 600. 126. Barrientos A, Volpini V, Casademont J, Genis D, Manzanares JM, Ferrer I, Corral J, Cardellach F, Urbano-Marquez A, Estivill X, Nunes V. A nuclear defect in the 4p16 region predisposes to multiple mitochondrial DNA deletions in families with Wolfram syndrome. J Clin Invest 1996;97:1570-1576. 127. Barrett TG, Scott-Brown M, Seller A, Bednarz A, Poulton K, Poulton J. The mitochondrial genome in Wolfram syndrome. J Med Genet 2000;37:463-466. 128. Domenech E, Gomez-Zaera M, Nunes V. Study of the WFS1 gene and mitochondrial DNA in Spanish Wolfram syndrome families. Clin Genet 2004;65:463-469. 129. Bespalova IN, Van Camp G, Bom SJH, Brown DJ, Cryns K, DeWan AT, Erson AE, Flothmann K, Kunst HPM, Kurnool P, Sivakumaran TA, Cremers CWRJ, Leal SM, Burmeister M, Lesperance MM. Mutations in the Wolfram syndrome 1 gene (WFS1) are a common cause of low frequency sensorineural hearing loss. Hum Mol Genet 2001;10:2501-2508. 130. Young TL, Ives E, Lynch E, Person R, Snook S, MacLaren L, Cater T, Griffin A, Fernandez B, Lee MK, King MC. Non-syndromic progressive hearing loss DFNA38 is caused by heterozygous missense mutation in the Wolfram syndrome gene WFS1. Hum Mol Genet 2001;10:2509-2514. 131. Pennings RJE, Bom SJH, Cryns K, Flothmann K, Huygen PLM, Kremer H, Van Camp G, Cremers CWRJ. Progression of low-frequency sensorineural hearing loss (DFNA6/14-WFS1). Arch Otolaryngol Head Neck Surg 2003;129:421-426. 132. Cryns K, Pfister M, Pennings RJE, Bom SJH, Flothmann K, Caethoven G, Kremer H, Schatteman I, Koln KA, Toth T, Kupka S, Blin N, Nurnberg P, Thiele H, Van de Heyning PH, Reardon W, Stephens D, Cremers CWRJ, Smith RJ, Van Camp G. Mutations in the WFS1 gene that cause low- frequency sensorineural hearing loss are small non-inactivating mutations. Hum Genet 2002;110:389-394. 133. Lesperance, MM. WFS1 Gene Mutation and Polymorphism Database. http://www.khri.med.umich.edu/research/lesperance_lab/lfsnhl.shtml. 2006. Ref Type: Electronic Citation 134. Pennings RJE, Huygen PLM, van den Ouweland JM, Cryns K, Dikkeschei LD, Van Camp G, Cremers CWRJ. Sex-related hearing impairment in Wolfram syndrome patients identified by inactivating WFS1 mutations. Audiol Neurootol 2004;9:51-62.

34 Chapter 2

DFNA8/12 (TECTA)

2.1

Van gen naar ziekte; DFNA8/12, een autosomaal dominant overervend komvormig perceptief gehoorverlies

Cor W.R.J. Cremers, Rutger F. Plantinga, Hannie Kremer. Nederlands Tijdschrift voor Geneeskunde 2007;151:531-534. Abstract An autosomal dominant inherited disorder known as DFNA8/12 causes mild-to- moderate/severe midfrequency or mild-to-severe progressive highfrequency sensorineural hearing impairment. The causative gene, TECTA, encodes Į- tectorin, the most important noncollagenous component of the tectorial membrane in the cochlea and the otolith membrane in the maculae of the vestibular system. Mutations in the zona pellucida domain of Į-tectorin cause midfrequency hearing impairment, whereas mutations in the zonadhesin domain cause progressive highfrequency hearing impairment. The intact hearing in the low and high frequencies may prohibit successful correction with a hearing aid. Chapter 2.1

De ziekte Een "komvormig" perceptief gehoorverlies dat al aanwezig is op kinderleeftijd is uitzonderlijk. In een deel van de gevallen betreft het meerdere familieleden. In het toonaudiogram is het gehoorverlies het grootste in de middenfrequenties 500-2000 Hz en het geringste voor de lagere en hogere tonen. Een dergelijke toonaudio- metrische curve heeft dan een komvorm, heel instructief ook wel "cookie-bite"- audiogram genoemd. Het is aannemelijk dat het om een aangeboren gehoorverlies gaat. Toch blijft het gehoorverlies lang onopgemerkt, omdat er in het hogere tonengebied een tamelijk normale gehoorgevoeligheid is en daar de spraak beter verstaan wordt. Zoals normaal horenden bij het verstaan in een zeer rumoerige omgeving om beter te horen uitwijken naar het gebied van de hoge tonen, zo geldt voor deze personen met een komvormig gehoorverlies dat zij niet anders kunnen.

Komvormig gehoorverlies kan geïsoleerd en onverklaard vóórkomen. Een autosomaal dominant overervende vorm is al bekend sinds de jaren vijftig en zestig van de vorige eeuw.1 2 Toen is de aandoening in families beschreven en is vastgesteld dat het gehoorverlies met de leeftijd toeneemt. In de jaren negentig van de vorige eeuw werd het mogelijk door in veelvuldig aangedane families genkoppelings- studies en daaropvolgend genidentificatie te verrichten om de niet-syndromale autosomaal dominant voorkomende vormen van slechthorendheid van elkaar te onderscheiden. De codering DFNA werd verkozen met een nummer als toevoeging om deze nieuw herkende ziektebeelden te benoemen. DFN staat voor "deafness" en de A staat voor "autosomaal dominant overervend". Het toegevoegde nummer geeft de volgorde in de tijd aan waarop de aandoening via genkoppeling geïdentificeerd werd. In dit geval bleken een Oostenrijkse en een Belgische familie met een middenfrequentieslechthorendheid, die eerder vanwege verschillende genkoppe- lingsresultaten afzonderlijk DFNA8 en DFNA12 benoemd waren, toch eenzelfde gebied voor genkoppeling en eenzelfde aangedane gen TECTA als grondslag te hebben.3 4 De benaming werd daardoor DFNA8/12.

Sommige families met DFNA8/12 laten gehoorverlies in met name de hoge tonen zien. Het gen voor DFNA8/12 werd TECTA genoemd, omdat bij deze ziekte de tectoriale membraan is aangedaan. Immers, het gen TECTA codeert voor Į-tectorine, dat de belangrijkste niet-collagene component van de tectoriale membraan in de cochlea en de otolietenmembraan in de maculae van het vestibulaire systeem is.

39 DFNA8/12 (TECTA)

De tectoriale membraan bestaat uit een extracellulaire matrix die over het orgaan van Corti heen ligt en in contact staat met de buitenste haarcellen (figuur 1). Deze tectoriale membraan speelt door de aandrijving van de haarcellen een belangrijke rol bij overdracht van geluid aan de haarcellen in de cochlea.5 Deze kennis maakt inzichtelijk waarom er bijzondere audiometrische bevindingen verwacht kunnen worden.6

Figuur 1. Illustratie van het binnenoor, met in detail een doorsnede van het Orgaan van Corti.

Bijzondere audiometrische bevindingen en de behandeling Op zichzelf is een komvormig audiogram al een bijzondere bevinding. Het vóórkomen daarvan binnen een familie wijst naar een erfelijke bepaaldheid en dan gaat het om een zich herhalende aandoening met eenzelfde onderliggende stoornis. Bijzonder is dat bij DFNA8/12 de geluidsoverdracht in het binnenoor verstoord is en wel voordat het eigenlijke zintuiglijke orgaan van Corti bereikt wordt. Het gaat daarmee om een geleidingsslechthorendheid in het binnenoor, zoals er ook bij een aandoening in het middenoor een geleidingsstoornis is.6 Een en ander betekent dat met hoortoestellen het probleem van onvoldoende geluidsoverdracht volledig

40 Chapter 2.1 gecorrigeerd kan worden om een verder intact zintuiglijk orgaan van Corti aan te sturen. Voorwaarde blijft dat het gehoorverlies in de hoge en lage tonen ook voldoende groot is om een hoortoestelaanpassing succesvol te kunnen laten zijn. Bij een dergelijk type binnenoor-gehoorverlies zal niet het bezwaar optreden dat de versterking van het geluid snel te sterk is en als te luid wordt ervaren.

Bij een licht tot 40 dB groot komvormig gehoorverlies met een bijna normaal gehoor in de hogere en lagere tonen kan een aanpassing van een hoortoestel mislukken door teveel hinder van omgevingsgeluiden (lage tonen) en een nog te goed verstaan in de hogere tonen. Een en ander betekent dat de revalidatiemogelijkheden vooral voor schoolgaande kinderen met DFNA8/12 veelal onvoldoende zijn om hen op- timaal te laten functioneren. Het gehoorverlies is in sommige families progressief. Multidisciplinaire begeleiding vanuit een kinderaudiologisch centrum is aange- wezen om elke patiënt optimaal te kunnen begeleiden.

Het gen Het TECTA-gen is gelegen op chromosoom 11q23.3 en bestaat uit 23 exonen. Het messenger-RNA is 6468 basenparen lang en codeert voor een eiwit, Į-tectorine, van 2155 aminozuren (Ensemble Human Genome; www.ensembl.org/homo_sapiens). Het gen komt alleen in het binnenoor hoog tot expressie. Het eiwit bestaat uit verschillende domeinen waaronder het zona-pellucida- en het zonadhesine-domein. Mutaties in TECTA-gen die het zona-pellucidadomein veranderen, veroorzaken een middenfrequentieslechthorendheid, terwijl mutaties die het zonadhesinedomein veranderen met name slechthorendheid van de hoge tonen veroorzaken (tabel).7 12 Verder blijken mutaties die een vervanging van cysteïne door een ander aminozuur veroorzaken, samen te gaan met een progressief beloop van het gehoorverlies.12 In figuur 2 zijn de gemiddelde audiogrammen van de beschreven families met niet- progressieve middenfrequentieslechthorendheid weergegeven.

Het eiwit en het orgaan van Corti Het TECTA-gen codeert voor D-tectorine, dat de belangrijkste niet-collagene com- ponent is van de tectoriale membraan in de cochlea en de otolietenmembraan in de maculae van het vestibulaire systeem. In het Į-tectorine kan een aantal bekende domeinen worden onderscheiden: het entactine G1-domein, het zonadhesine- domein met "repeats" van de von-willebrand-factor type D en het zonapellucida- domein.13

41 DFNA8/12 (TECTA)

Tabel 1. TECTA-genmutaties bij slechthorendheid type DFNA8/12 en kenmerken van het betreffende fenotype. Mild gehoorverlies = 20-40 dB, matig ernstig 41-70 dB en ernstig = 71- 95 dB.

Land van Mutatie* Domein Leeftijd van Fenotype Aangedaan Frequenties herkomst ontstaan

Frankrijk7 C1619S ZA Variabel Mild tot matig ernstig Progressief Hoog

Zweden8 C1057S ZA Postlinguaal Mild tot ernstig Progressief Hoog

Turkije9 C1509G ZA Prelinguaal Mild tot matig ernstig Progressief Hoog

Spanje10 C1837G ZP Postlinguaal Mild tot matig ernstig Progressief Midden

Oostenrijk3 Y1870C ZP Prelinguaal Matig ernstig Stabiel Midden

L1820F, België4 ZP Prelinguaal Matig ernstig Stabiel Midden G1824D

Japan11 R2021H ZP Prelinguaal Mild tot matig ernstig Stabiel Midden

Nederland12 R1890C ZP Prelinguaal Mild tot matig ernstig Stabiel Midden

*"C1619S" betekent dat op plaats 1619 in het eiwit het aminozuur cysteïne (C) is vervangen door serine (S); D = asparaginezuur; F = fenylalanine; G = glycine; H = histidine; L = leucine; R = arginine; Y = tyrosine

Mutaties in het TECTA-gen die leiden tot de vervanging van een cysteïne door een ander aminozuur of die leiden tot een extra cysteïne, veranderen wellicht de zwavelbruggen in het D-tectorine en daardoor de structuur. Samen met drie verschillende typen collagenen en de twee andere niet-collagene glycoproteïnen E- tectorine en otogeline, vormt D-tectorine de extracellulaire matrix van de tectoriale membraan. De stereocilia van de buitenste haarcellen maken contact met de tectoriale membraan, die van de binnenste haarcellen niet direct. De precieze functie van de tectoriale membraan begint langzaam duidelijk te worden. Defecten in deze membraan leiden tot gehoorverlies, zoals DFNA13 (COL11A2) en DFNA8/12. Mutaties in COL11A2 leiden tot een abnormale type XI-collageen-D2-keten en daardoor tot disorganisatie van de collageenfibril (of type II-collageen) in de tectoriale membraan.

42 Chapter 2.1

dB HL -10 0 20

40 60

80

100

120

.25 .5 1 248kHz

Figuur 2. Gemiddeld audiogram van de beschreven families met niet-progressieve middenfrequentie slechthorendheid:, Oostenrijk3;  , België4; ------, Japan11; ——, Nederland12.

Onderzoek aan muizenmodellen met een defect in het TECTA-gen heeft aangetoond dat de tectoriale membraan essentieel is voor het positioneren van stereocilia van de buitenste haarcellen, dusdanig dat een bepaalde frequentie in de betrokken regio van de cochlea optimaal versterkt wordt.14 Ook werd duidelijk dat de tectoriale membraan essentieel is voor de beweging van de basale membraan die ervoor zorgt dat de binnenste haarcellen geactiveerd worden bij specifieke frequenties.14 Muizen waarbij D-tectorine ontbreekt, hebben een gehoorverlies van 35 dB. De structuur van de tectoriale membraan is abnormaal en het ligt niet over het orgaan van Corti heen. Otoakoestische emissies werden niet waargenomen bij deze muizen.5

43 DFNA8/12 (TECTA)

De populatie Binnen de patiëntenpopulatie van een kinderaudiologisch centrum is komvormig perceptief gehoorverlies weliswaar zeldzaam, maar niet ongewoon. Bij een belangrijk deel van de patiënten gaat het om een onverklaard geïsoleerd vóór- komen. In de overige gevallen blijken meerdere familieleden aangedaan. Omdat een hoortoestel niet steeds uitkomst brengt, blijft het gehoorverlies vaak voor de omgeving verborgen. Het vóórkomen van komvormig gehoorverlies bij andere familieleden valt daarom niet steeds op. Kleine families met meerdere aangedane leden zijn bij een kinderaudiologisch centrum niet zeer zeldzaam. Wij schatten dat er bij het Nijmeegs audiologisch centrum enkele tientallen families met deze aandoening bekend zijn. Grote families met veel (10-15) aangedane leden zijn wel tamelijk zeldzaam. Inmiddels is het gelukt om 4 families met meer dan 10 aan- gedane personen in kaart te brengen. Met behulp van dergelijke families kan via koppelingsonderzoek naar het type DFNA gezocht worden. Komvormig gehoor- verlies kan, meer incidenteel dan systematisch zoals bij DFNA8/12, ook vóórkomen bij DFNA13.

Diagnostiek Audiometrisch onderzoek binnen een familie maakt duidelijk of er ook bij andere familieleden komvormig perceptief gehoorverlies bestaat. Een autosomaal dominante overerving kan daarmee aannemelijk worden. Mutatieanalyse van het TECTA-gen wordt nog niet aangeboden als routine-DNA-diagnostiek. In het oto- genetisch researchlaboratorium te Nijmegen is dit onderzoek wel mogelijk. Er is om redenen van efficiëntie een voorkeur om bij voldoende omvang van de aangedane familie koppelingsonderzoek voor de chromosomale regio van het TECTA-gen te verrichten om vooraf te bevestigen dat het gaat om DFNA8/12. Dergelijk gen- koppelingsonderzoek voor een familie kan in elk genetisch centrum uitgevoerd worden. De Nijmeegse otogenetische researchgroep is, indien gewenst, bereid vooraf het benodigde klinische familieonderzoek te verrichten.

44 Chapter 2.1

Referenties 1. Williams F, Lyons JP, Stein SP, Roblee L. Progressive hereditary nerve deafness; report of four cases in one family. Med Ann Dist Columbia 1954;23:681-683. 2. Martensson B. Dominant hereditary nerve deafness. Acta Otolaryngol 1960;52:270-274. 3. Kirschhofer K, Kenyon JB, Hoover DM, Franz P, Weipoltshammer K, Wachtler F, et al. Autosomal-dominant, prelingual, nonprogressive sensorineural hearing loss: localization of the gene (DFNA8) to chromosome 11q by linkage in an Austrian family. Cytogenet Cell Genet 1998;82:126-130. 4. Verhoeven K, Van Laer L, Kirschhofer K, Legan PK, Hughes DC, Schatteman I, et al. Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat Genet 1998;19:60-62. 5. Legan PK, Lukashkina VA, Goodyear RJ, Kossi M, Russell IJ, Richardson GP. A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 2000;28:273-285. 6. Plantinga RF, Cremers CWRJ, Huygen PLM, Kunst HPM, Bosman AJ. Audiological Evaluation of Affected Members From a Dutch DFNA8/12 (TECTA) Family. J Assoc Res Otolaryngol 2007;8: 1-7. 7. Alloisio N, Morlé L, Bozon M, Godet J, Verhoeven K, Van Camp G, et al. Mutation in the zonadhesin-like domain of a-tectorin associated with autosomal dominant non-syndromic hearing loss. Eur J Hum Genet 1999;7:255-258. 8. Balciuniene J, Dahl N, Jalonen P, Verhoeven K, Van Camp G, Borg E, et al. Alpha-tectorin involvement in hearing disabilities: one gene--two phenotypes. Hum Genet 1999;105:211-216. 9. Pfister MHF, Thiele H, Van Camp G, Fransen E, Apaydin F, Aydin O, et al. A genotype-phenotype correlation with gender-effect for hearing impairment caused by TECTA mutations. Cell Physiol Biochem 2004;14:369-376. 10. Moreno Pelayo MA, del Castillo I, Villamar M, Romero L, Hernandez Calvin FJ, Herraiz C, et al. A cysteine substitution in the zona pellucida domain of a-tectorin results in autosomal dominant, postlingual, progressive, mid frequency hearing loss in a Spanish family. J Med Genet 2001;38:E13. 11. Iwasaki S, Harada D, Usami S, Nagura M, Takeshita T, Hoshino T. Association of clinical features with mutation of TECTA in a family with autosomal dominant hearing loss. Arch Otolaryngol Head Neck Surg 2002;128:913-917. 12. Plantinga RF, de Brouwer AP, Huygen PLM, Kunst HPM, Kremer H, Cremers CWRJ. A Novel TECTA Mutation in a Dutch DFNA8/12 Family Confirms Genotype-Phenotype Correlation. J Assoc Res Otolaryngol 2006;7:173-181. 13. Legan PK, Rau A, Keen JN, Richardson GP. The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system. J Biol Chem 1997;272:8791-8801. 14. Legan PK, Lukashkina VA, Goodyear RJ, Lukashkin AN, Verhoeven K, Van Camp G, et al. A deafness mutation isolates a second role for the tectorial membrane in hearing. Nat Neurosci 2005;8:1035-1042.

45

2.2

A novel TECTA mutation in a Dutch DFNA8/12 family confirms genotype-phenotype correlation

Rutger F. Plantinga*, Arjan P.M. de Brouwer*, Patrick L.M. Huygen, Henricus P.M. Kunst, Hannie Kremer, Cor W.R.J. Cremers. Journal of the Association for Research in Otolaryngology 2006; 7: 173-181.

* Both authors contributed equally Abstract A novel TECTA mutation, p.R1890C, was found in a Dutch family with non- syndromic autosomal dominant sensorineural hearing impairment. In early life, presumably congenital, hearing impairment occurred in the midfrequency range, amounting to about 40 dB at 1 kHz. Speech recognition was good with all phoneme recognition scores exceeding 90%. An intact horizontal vestibulo-ocular reflex was found in four tested patients. The missense mutation is located in the zona pellucida (ZP) domain of D-tectorin. Mutations affecting the ZP domain of D- tectorin are significantly associated with midfrequency hearing impairment. Substitutions affecting other amino acid residues than cysteines show a significant association with hearing impairment without progression. Indeed, in the present family progression seemed to be absent. In addition, the presently identified mutation affecting the ZP domain resulted in a substantially lesser degree of hearing impairment than was previously reported for DFNA8/12 traits with mutations affecting the ZP domain of D-tectorin. Chapter 2.2

Introduction Various phenotypes of autosomal dominant, sensorineural, nonsyndromic hearing impairment can be distinguished. Up untill now, 54 loci have been mapped and 21 genes have been identified.1. The different gene loci for these nonsyndromic types of hearing impairment have been designated DFN (DeaFNess) and are numbered in chronological order of discovery. Autosomal dominant types are referred to as DFNA. These types of hearing impairment can be characterised by age of onset, presence and degree of progression, severity of hearing impairment and audiometric configuration. One of these configurations is the “cookie-bite”, U-shaped or trough- shaped audiometric configuration of midfrequency hearing impairment. This phenotype can be found in DFNA8/12 (TECTA) and DFNA13 (COL11A2).1.

DFNA8/12 has been mapped to the chromosomal locus 11q22-242,3 and is caused by mutations in TECTA.4,5 This gene encodes D-tectorin, the most important non- collagenous component of the tectorial membrane in the cochlea and the otolith membrane in the maculae of the vestibular system. The tectorial membrane consists of an extracellular matrix overlying the organ of Corti that contacts the outer cochlear hair cells and plays an important role in intracochlear sound transmission by ensuring optimal cochlear feedback.6. Recently, Legan et al.7 described a second major role for the tectorial membrane. Neural tuning curves relating to inner hair cell stimulation showed a remarkable loss in sensitivity in mutant mice. This effect was attributed to a loss in coupling of the inner hair cell bundles to tectorial membrane motion caused by enlargement of the subtectorial space in the region of these hair cells.

In this study we report a Dutch family with nonprogressive, presumably prelingual, midfrequency hearing impairment caused by a novel missense mutation affecting the zona pellucida (ZP) domain of D-tectorin. Statistical testing was performed by using this mutation and the mutations previously identified to show the significance of the association between the type of mutation and the respective phenotype.

Patients and methods A three-generation pedigree was established for the present family. Twenty-four family members participated in this study. Their medical history was taken and

49 Dutch DFNA8/12 (TECTA) family otologic examination was performed. Nonhereditary causes of hearing loss were excluded and written informed consent was obtained. All individuals included in this study underwent ; speech audiometry was only performed in affected persons. Vestibular function was tested in four patients. Blood samples were obtained for linkage analysis from 11 affected and 13 unaffected family members.

Audiometric analysis Pure tone and speech audiometry were performed in a sound treated room, conforming to the International Standards Organisation (ISO) standards.8,9 The individual 95th percentile threshold values of presbyacusis (P95) in relation to the patient’s sex and age were derived for each frequency by using the ISO 7029 method.10 Individuals were considered affected if the best hearing ear showed thresholds beyond the P95.

Speech audiometry Speech audiometry was performed in a quiet environment using standard monosyllabic Dutch word lists. The maximum monaural phoneme score (% correct recognition) was derived from a performance vs. intensity plot.

Vestibulo-ocular examination Vestibulo-ocular responses were evaluated in four patients (aged 23, 39, 43 and 45 years) by using electronystagmography (in the dark with open eyes) with computer analysis as previously described.11 The horizontal vestibulo-ocular reflex (VOR) was evaluated by performing velocity-step tests at 90º/s. Ocular motor evaluation comprised saccades, smooth pursuit eye movements and optokinetic , as well as gaze-evoked and spontaneous nystagmus.

Genotyping DNA from lymphocytes was isolated as described by Miller et al.12 Polymorphic microsatellite markers were amplified by using 50 ng genomic DNA in 20 mM Tris-

HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 2 U Taq Polymerase (Invitrogen, Breda, the Netherlands), 125 µM dATP, 125 µM dGTP, 125 µM dTTP, 1.5 µM dCTP, 400 nM 32P- dCTP (111 TBq/mmol; MP Biomedicals, Irvine, CA, USA), 2.7 ng forward primer, and 2.7

50 Chapter 2.2 ng reverse primer in a total volume of 25 µl. PCR products were analysed as described by Kremer et al.13

Linkage analysis Two-point lod scores and the maximum lod score were calculated with the Mlink and Ilink subroutines, respectively, of the LINKAGE programme (version 5.1).14,15 Full penetrance was assumed and the disease allele frequency was set at 0.0001. The phenocopy rate was estimated to be 1 in 1,000.

Mutation analysis Primer sequences and conditions for amplification of all exons of TECTA (GenBank ID NM_005422.1) are available on request. PCR products were sequenced by using the ABI PRISM BigDye Terminator Cycle Sequencing V2.0 Ready Reaction Kit and analysed with the ABI PRISM 3730 DNA analyser (Applied Biosystems, Foster City, CA, USA). Of the last exon, 450 nucleotides of the 3’-untranslated region were analysed. Segregation in the family and presence of the c.248C>T nucleotide change in 165 controls was tested by amplification of exon 3 and subsequent digestion with NlaIII according to the manufacturer’s protocols (Invitrogen). Segregation in the family and presence of the nucleotide substitution, c.5668C>T, in 184 controls was tested by amplification of exon 18 and subsequent digestion with BsgI according to the manufacturer’s protocols (Invitrogen).

Data analysis Binaural mean threshold levels were used to perform cross-sectional linear regression analysis (threshold on age) at each frequency. The presence/absence of progression was assessed by testing whether or not the 95% confidence interval for the regression coefficient (slope) included zero. Phoneme recognition scores (binaural mean) were plotted against the degree of impairment, expressed as the binaural mean pure tone average (PTA) at the frequencies 1, 2 and 4 kHz (PTA 1, 2, 4 kHz). Curve fitting in this performance-impairment plot was conducted according to a previously reported method using a sigmoidal equation with variable slope.16 A previously described group of subjects with only presbyacusis17 was used as a reference group. Only those subjects with presbyacusis who had a matching degree of impairment by PTA 1, 2, 4 kHz were used for statistical testing. A 2 x 2 contingency table was constructed by using one of the fitted curves as a dividing line for dichotomising the speech recognition scores in both groups and Fisher’s exact test

51 Dutch DFNA8/12 (TECTA) family was performed. Contingency tables (2 x 2) were also used to test for the possible significance (p < 0.05 in Fisher’s exact test) of associations between genotype and phenotype features.

Results The pedigree (Figure 1) comprised three generations and included 14 affected family members (8 males and 6 females), 11 of whom were still alive and willing to participate in this study. The pattern of inheritance is autosomal dominant.

Figure 1. Pedigree and chromosome 11 haplotypes of the family. The at-risk haplotype is indicated by the black bar. If the phase is unknown, the haplotype is marked with a thin line. Brackets indicate deduced marker alleles. The marker order is according to the Human Genome Working Draft May 2004 and corresponds to the deCODE genetic map.18

There was no evidence of any other cause of hearing impairment, except for one patient who had undergone stapes replacing surgery for unilateral . This patient was included in the present study by only using audiometric data from her other ear. The first symptoms of hearing impairment were reported at ages ranging from <1 to 30 years. Vestibular symptoms were not reported. Otoscopy was normal

52 Chapter 2.2 in all persons except the patient mentioned above. Pure tone audiograms (Figure 2) were symmetric and often showed a so-called cookie-bite shape, which indicates that predominantly the middle frequencies are affected. The highest threshold was most often found at 1 kHz, followed by 2 kHz. It was usually in the range of 40-60 dB, apparently independent of age.

dB Pat06 dB Pat 08 dB Pat01 dB Pat09 -10 -10 -10 -10 0 5.87 0 6.67 0 6.21 0 14.46 6.33 6.35 20 20 20 20 6.83 40 40 40 7.29 40 60 60 60 11.36 60

80 80 80 11.56 80 13.14 100 100 100 100

120 120 120 120 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz

dB Pat03 dB Pat04 dB Pat11 dB Pat10 -10 -10 -10 -10 0 22.7 0 7.84 0 28.82 0 18.08 8.58 37.57 20 20 20 20 9.5 40 40 40 40 13.94 60 60 17.73 60 60

80 80 24.94 80 80

100 100 100 100

120 120 120 120 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz

dB Pat02 dB Pat07 dB Pat05 -10 -10 -10 0 35.87 0 18.55 0 21.3 26.76 20 43.2 20 44.76 20 35.79 40 40 40 47.3 60 60 60

80 80 80

100 100 100

120 120 120 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz

Figure 2. Binaural mean air conduction thresholds of the affected family members shown in audiogram format. Numbers designate age in years. Dotted line represents P50 thresholds for the last age >20 years according to ISO 7029.10 The panels are ordered by age at last complete audiogram.

Audiometric analysis All available cross-sectional data, combining individual longitudinal measurements (seven cases), i.e. only those pertaining to the patient’s last complete audiogram, and single-snapshot measurements (four cases) are shown for the separate frequencies in figure 3.

53 Dutch DFNA8/12 (TECTA) family

0.25 kHz 0.5 kHz 120 120

100 100

80 80

60 60

40 40

20 20

0 0 0 10 20 30 40 50 0 10 20 30 40 50 1 kHz 2 kHz 120 120

100 100

80 80

60 60

40 40

20 20

0 0 0 10 20 30 40 50 0 10 20 30 40 50 4 kHz 8 kHz 120 120

100 100

80 80

60 60

40 40

20 20 Threshold (dB HL) (dB Threshold 0 0 0 10 20 30 40 50 0 10 20 30 40 50 Age (y)

Figure 3. Cross-sectional linear regression analysis of the patient’s binaural mean thresholds at 0.25-8 kHz, as measured in the last complete audiological test. Numbers designate age in years.

We found only shallow positive or negative slopes. Indeed, the 95% confidence interval for the slope of the regression line included zero at all frequencies. Thus, it appeared that the thresholds did not depend on age. Age could therefore be ignored and mean thresholds covering all ages were calculated (Figure 4).

54 Chapter 2.2

dB HL -10 0 20

40 60

80

100

120

.25 .5 1 2 4 8 kHz

Figure 4. Mean threshold ± 1 across-subjects SD for the present family (open circles and fat solid line). Mean thresholds for previously reported nonprogressive DFNA8/12 traits are included:, Kirschhofer et al.2;  , Govaerts et al.5; ------, Iwasaki et al.19.

Figure 4 includes the mean evaluable thresholds previously reported for other DFNA8/12 families with nonprogressive hearing loss.2,5,19 Remarkably, the mean threshold for the present family is substantially lower than the mean threshold of any of the other families. The difference amounts to 20-30 dB. The previously described families showed a tendency for a maximum threshold at 2 kHz rather than 1 kHz in the present family.

Speech recognition All of the present patients had maximum phoneme recognition scores of >90% at a

PTA1,2,4 kHz of 60 dB or better (Figure 5). The presbyacusis data used as a reference were matched by selecting the subjects with a PTA1,2,4 kHz of 60 dB or better. Fischer’s exact test indicated a significant difference in favour of the present patients (p=0.018 or 0.034, depending on the dividing line). As can be read from the fitted curves, the difference amounted to about 10-15% on average at a PTA1, 2, 4 kHz close to 60 dB (Figure 5).

55 Dutch DFNA8/12 (TECTA) family

100

80

60

40

20 %Recognition 0 10 20 30 40 50 60

PTA1,2,4 kHz (dB HL)

Figure 5. Binaural mean phoneme recognition score (% correct) plotted against binaural mean PTA1,2,4 kHz for patients of the present family (open circles and bold solid line) and for a group of subjects with presbyacusis showing a matching degree of impairment (crosses and thin solid line13).

Vestibulo-ocular examination None of the patients had any vestibular symptoms. Ocular motor tests were normal in the four patients tested. Intact vestibular function was found with a normal horizontal VOR gain in all of these patients, with an abnormally long dominant VOR time constant in two of them.

Linkage analysis The two DFNA loci associated with midfrequency hearing impairment, DFNA8/12 and DFNA13, were tested for linkage with polymorphic markers flanking or within the causative genes TECTA and COL11A2, respectively. For the DFNA8/12 locus, linkage was detected with a maximum lod score of 4.00 (ș = 0.0) for the intragenic marker D11S4167. The DFNA13 locus was excluded (lod score <-2.0) by using the

56 Chapter 2.2 markers D6S1666, D6S273, D6S276, and D6S1615. The at-risk haplotype, which includes the DFNA8/12 locus on chromosome 11, is depicted in figure 1.

DNA sequencing of TECTA DFNA8/12 is caused by missense mutations in TECTA. We analysed TECTA for mutations in all 23 exons and intron-exon boundaries in family member III-11. Both the forward and reversed DNA sequence of exon 3 showed a nucleotide change (c.248C>T) that is predicted to result in the substitution of a methionine for a threonine at position 83 (Figure 6A).

Figure 6. Nucleotide changes in exon 3 and exon 18 of TECTA. Chromatograms showing the c.248C>T transversion in exon 3 in individual III:11 (A), and the normal DNA sequence in a healthy individual (B). Chromatograms showing the c.5668C>T transversion in exon 18 individual III:11 (C), and the normal DNA sequence in a healthy individual (D).

This novel variant removes an NlaIII restriction site. The substitution segregated with the disease and was not present in 330 alleles from healthy controls as determined by restriction analysis (data not shown). In addition, another change, c.5668C>T, was detected that is predicted to result in the substitution of a cysteine for an arginine at position 1890 in the ZP domain of Į-tectorin (Figure 6C). This

57 Dutch DFNA8/12 (TECTA) family change creates a BsgI restriction site, segregated with the disease and was not present in 368 alleles from healthy controls (data not shown). Finally, four known single nucleotide polymorphisms (rs612969, rs536069, rs520805, and rs586473; www.ncbi.nlm.nih.gov/SNP) were identified in family member III-11.

Genotype-phenotype correlation Alpha-Tectorin is composed of three distinct modules: the entactin G1 domain, the zonadhesin (ZA) domain with von Willebrand factor type D repeats, and the ZP domain.20 Mutations affecting the ZP domain are associated with midfrequency hearing impairment, whereas mutations in the ZA domain are associated with hearing impairment primarily affecting the high frequencies (table 1).2,4,5,19,21-24 Hearing impairment can range from mild to severe and has prelingual or postlingual onset. Furthermore, mutations causing substitution of cysteine residues are associated with progressive hearing impairment.

Table 1: DFNA8/12 TECTA mutations and the associated phenotypes

Origin Mutation Exon Domain Onset Phenotype* Frequency

21 French C1619S 14 ZA Variable Mild to moderate-severe, progressive High 22 Swedish C1057S 10 ZA Postlingual Mild to severe, progressive High 23 Turkish C1509G 13 ZA Prelingual Mild to moderate, progressive High 24 Spanish C1837G 17 ZP Postlingual Mild to moderate, progressive Mid 2 Austrian Y1870C 18 ZP Prelingual Moderate, stable Mid 4-5 Belgian L1820F 17 ZP Prelingual Moderate, stable Mid G1824D 19 Japanese R2021H 20 ZP Prelingual Mild to moderate, stable Mid Dutch R1890C 18 ZP Prelingual Mild to moderate, stable Mid *According to the GENDEAF criteria: mild, 20-40 dB; moderate, 41-70 dB; severe, 71-95 dB.25

Two-by-two contingency tables were used for testing the possible significance (p <0.05 in Fisher’s exact test) of associations between the position and nature of the amino acid substitutions and the specific phenotypic features (table 2). Mutations in the ZP domain are significantly associated with midfrequency hearing impairment, whereas mutations in the ZA domain are associated with high

58 Chapter 2.2 frequency hearing loss (p=0.018). Cysteine replacing mutations are associated with progressive hearing impairment (p=0.014).

Table 2: Two-by-two contigency showing association between the type of mutation (geno- type) and the phenotype. Values within parentheses pertain to the number of observations prior to the inclusion of the present study. A Affected domain Mid frequency type High frequency type ZP 5 (4) 0 ZA 0 3 Fisher’s exact test: p=0.018 (0.029).

B C-any substitution Stable Progressive Yes 0 4 No 4 (3) 0 Fisher’s exact test: p=0.014 (0.029).

Discussion Two changes, p.R1890C and p.T83M, were detected in D-tectorin in a family with autosomal dominant midfrequency hearing impairment. The mutation p.R1890C is present in the ZP domain of Į-tectorin, whereas p.T83M is not found in a specific domain. Both amino acid residues are conserved among human, rat, and chicken Į- tectorin. The identity between these three orthologs is higher than 70%. Considering the phenotype and the position of the mutation, p.R1890C is likely to be causative for hearing impairment in the family under study, because cysteines in the ZP domain are involved in the intra- and/or intermolecular interactions of Į-tectorin, and mutations of cysteines or other amino acid residues into cysteines have been shown to cause autosomal dominant hearing impairment, due to problems with Į- tectorin secretion.26 Also, the p.T83M change seems to be a relatively mild amino acid substitution according to the Dayhoff table.27 The nucleotide change is not predicted to affect splicing of the TECTA mRNA. However, because this change was not present in controls, it can not be excluded that it has an effect on the phenotype of these patients or it may even act synergistically with the p.R1890C mutation.

59 Dutch DFNA8/12 (TECTA) family

It has been suggested in a previous report that patients with TECTA-based mid- frequency hearing impairment might be less prone to presbyacusis because they are generally exposed to lower-than-usual levels of sound energy at the level of the organ of Corti.2 Unaided phoneme recognition scores were all > 90% and proved to be significantly higher than scores found in a reference group of patients with presbyacusis. The difference amounted to 10-15% at around a PTA1, 2, 4 kHz of 60 dB. It is likely that this difference in favour of the present patients relates to the fact that they have substantially better thresholds at higher frequencies than the subjects with presbyacusis. If a protective effect against presbyacusis indeed occurs, it might be less important in the present family, given the observation that the threshold levels at 4-8 kHz were about 20-30 dB, whereas they amounted to 40-60 dB in the previously reported families (Figure 4). Nevertheless, we have attempted to substantiate the presence of any such protective effect by correcting the individual 10 thresholds at each frequency for the median (P50) threshold indicated by ISO 7029. Linear regression analysis (threshold on age) similar to the one illustrated in figure 3 performed on the corrected thresholds produced plots with regression lines that mainly showed negative slopes. However, none of these slopes was significantly negative (data not shown) and thus did not support any hypotheses about protection from presbyacusis.

Differences in phenotype among DFNA8/12 families appear to be related to the position of the mutations in either the ZP or ZA domain of D-tectorin as well as the nature of the amino acid substitution (Table 1). Mutations in the ZP and ZA domains have been related to midfrequency and high frequency hearing impairment, respectively.2,4,5,19,21-24 Substitutions replacing cysteines have been implicated in progressive hearing impairment.21-24 There are now sufficient observations to show the significance of these genotype-phenotype correlations (Table 2A, B).

There is a significant association between mutations affecting the ZP domain and the midfrequency impairment phenotype as well as between mutations affecting the ZA domain and the high frequency impairment phenotype (Table 2A). There is also a significant association between mutations causing cysteine-replacing substitutions and age-related progression of the hearing impairment (Table 2B). It should be noted that the level of significance (0.05 as usual) might be adjusted for multiple testing as two independent tests were performed with the same material. This would require the p value to be <0.0253 instead of <0.05. In Table 2, the p values

60 Chapter 2.2 fulfilled this requirement; however, the collective data prior to the inclusion of the present family (values in parentheses in Table 2A, B) did not. Thus, the data on TECTA genotypes and the corresponding phenotypes support the previously suggested associations on the basis of significant test results.

In the four patients, we found an intact horizontal VOR. Goodyear and Richardson28 found abundant expression of Į-tectorin in utricular and saccular otoconial membrane, not in the crista ampullaris. Legan et al6 found substantially reduced otoconial membranes with only a few, abnormally large, scattered otoconia in mice homozygous for a targeted deletion in Į-tectorin; the cupulae in the semicircular canals were normal. These mice did not show any spontaneous vestibular behavioural deficit, such as circling or head bobbing. However, no righting or swimming tests were performed. The present finding that the horizontal VOR was intact in the present patients conforms with the previous finding of Iwasaki et al.19, i.e. the only other study in which vestibular testing was performed. However, otolith reflexes are not covered by the type of vestibular testing performed. A suggestive anamnestic finding was reported for a French family whose three affected members had started walking only at 24 months of age.21 Vestibular testing was not performed in this family. It seems that the question as to whether DFNA8/12 patients have normal or abnormal otolith reflexes still requires appropriate instrumental otolith-testing paradigms. Given the type of histopathology observed in animal models for TECTA6, imaging of the inner ear cannot be expected to contribute to the knowledgepool as yet. Indeed, no abnormal scanning results have been obtained up to now.

In summary, we identified a novel mutation, p.R1890C, in the ZP domain of D- tectorin in a family with a somewhat milder degree of midfrequency hearing impairment than previously reported to be associated with mutations in this domain. Statistical tests on genotype-phenotype correlations of the present and collective data on DFNA8/12 have now revealed a significant association between the affected Į-tectorin domain (ZP or ZA) and the type of hearing impairment (mid- frequency or high frequency type), as well as a significant association between a cysteine-replacing substitution and age-related progression in hearing impairment.

61 Dutch DFNA8/12 (TECTA) family

References 1. Van Camp G, Smith RJH. Hereditary Hearing Loss Homepage. World Wide Web URL: http://webhost.ua.ac.be/hhh/ Accessed July 2005. 2. Kirschhofer K, Kenyon JB, Hoover DM, et al. Autosomal-dominant, prelingual, non-progressive sensorineural hearing loss: localization of the gene (DFNA8) to chromosome 11q by linkage in an Austrian family. Cytogenet Cell Genet 1998;82:126-30. 3. Verhoeven K, Van Camp G, Govaerts PJ, et al. A gene for autosomal dominant non-syndromic hearing loss (DFNA12) maps to chromosome 11q22-24. AM J Human Genet 1997;60:1168-1173. 4. Verhoeven K, Van Laer L, Kirschhofer K, et al. Mutations in the human Į-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat Genet 1998;19:60-2. 5. Govaerts PJ, De Ceulaer G, Daemers K, et al. A new autosomal-dominant locus (DFNA12) is responsible for a non-syndromic, mid-frequency, prelingual and non-progressive sensorineural hearing loss. Am J Otol 1998;19:718-23. 6. Legan PK, Lukashkina VA, Goodyear RJ, et al. A targeted deletion in Į-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 2000;28:273-85. 7. Legan PK, Lukashkina VA, Goodyear RJ, et al. A deafness mutation isolates a second role for the tectorial membrane in hearing. Nat Neurosci 2005;8:1035-42. 8. ISO 389. Acoustics. Standard reference zero for the calibration of pure tone air conduction audiometers. Geneva: International Organisation for Standardization, 1985. 9. ISO 8253-1. Acoustics. Audiometric test methods, I: basic pure tone air and bone conduction threshold audiometry. Geneva: International Organisation for Standardization, 1989. 10. ISO 7029. Acoustics. Threshold as hearing by air conduction as a function of age and sex for otologically normal persons. Geneva: International Organisation for Standardization, 1984. 11. Verhagen WIM, ter Bruggen JP, Huygen PLM. Oculomotor, auditory, and vestibular responses in myotonic dystrophy. Arch Neurol 1992;49:954-96. 12. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. 13. Kremer H, Pinckers A, van den Helm B, et al. Localization of the gene for dominant cystoid macular dystrophy on chromosome 7p. Hum Mol Genet 1994;3:299-302. 14. Lathrop GM, Lalouel JM. Easy calculations of lod scores and genetic risks on small computers. Am J Hum Genet 1984;36:460-465. 15. Lathrop GM, Lalouel JM, White RL. Construction of human linkage maps: likelihood calculations for multilocus linkage analysis. Genet Epidemiol 1986;3:39-52. 16. Bom SJH, De Leenheer EMR, Lemaire FX, et al. Speech recognition scores related to age and degree of hearing impairment in DFNA2/KCNQ4 and DFNA9/COCH. Arch Otolaryngol Head Neck Surg 2001;127:1045-1048. 17. De Leenheer EMR, Huygen PLM, Coucke PJ, et al. Longitudinal and cross-sectional phenotype analysis in a new, large Dutch DFNA2/KCNQ4 family. Ann Otol Rhinol Laryngol 2002;111:267-74. 18. Kong A, Gudbjartsson DF, Sainz J, et al. A high-resolution recombination map of the human genome. Nat Genet 2002;31:241-247. 19. Iwasaki S, Harada D, Usami S, et al. Association of clinical features with mutation of TECTA in a family with autosomal dominant hearing loss. Arch Otolaryngol Head Neck Surg 2002;128:913-7.

62 Chapter 2.2

20. Legan PK, Rau A, Keen JN, et al. The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system. J Biol Chem 1997;272:8791-801. 21. Alloisio N, Morlé L, Bozon M, et al. Mutation in the zonadhesin-like domain of Į-tectorin associated with autosomal dominant non-syndromic hearing loss. Eur J Hum Genet 1999;7:255-8. 22. Balciuniene J, Dahl N, Jalonen P, et al. Alpha-tectorin involvement in hearing disabilities: one gene - two phenotypes. Hum Genet 1999;105:211-6. 23. Pfister M, Thiele H, Van Camp G, et al. A genotype-phenotype correlation with gender-effect for hearing impairment caused by TECTA mutations. Cell Physiol Biochem 2004;14:369-76. 24. Moreno Pelayo MA, del Castillo I, Villamar M, et al. A cysteine substitution in the zona pellucida domain of Į-tectorin results in autosomal dominant, postlingual, progressive, mid frequency hearing loss in a Spanish family. J Med Genet 2001;38:E13 25. Mazzoli M, Van Camp G, Newton V, et al. Recommendations for the description of genetic and audiological data for families with non-syndromic hereditary hearing impairment. Hereditary Hearing Loss Homepage (see hyperlink in ref 1) 2005. 26. Jovine L, Qi H, Williams Z, et al. The ZP domain is a conserved module for polymerization of extracellular proteins. Nat Cell Biol 2002;4:457-61. 27. Dayhoff MO, Schwart RM, Orcutt BC. A model of evolutionary change in proteins. In: Dayhoff, M. (Ed.), Atlas of Protein Sequence and Structure, vol. 5 (1978). National Biomedical Research Foundation, Washington DC, 345-352. 28. Goodyear RJ, Richardson GP. Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structual diversity. J Neurobiol 2002;52:212-27.

63

2.3

Audiological evaluation of affected members from a Dutch DFNA8/12 (TECTA) family

Rutger F. Plantinga, Cor W.R.J. Cremers, Patrick L.M. Huygen, Henricus P.M. Kunst, Arjan J. Bosman. Journal of the Association for Research in Otolaryngology 2007;8:1-7. Abstract In DFNA8/12, an autosomal dominantly inherited type of nonsyndromic hearing impairment, the TECTA gene mutation causes a defect in the structure of the tectorial membrane in the inner ear. Because DFNA8/12 affects the tectorial membrane, patients with DFNA8/12 may show specific audiometric characteristics. In this study, five selected members of a Dutch DFNA8/12 family with a TECTA sensorineural hearing impairment were evaluated with pure-tone audiometry, loudness scaling, speech perception in quiet and noise, difference limen for frequency, acoustic reflexes, otoacoustic emissions, and gap detection. Four out of five subjects showed an elevation of pure tone thresholds, acoustic reflex thresholds, and loudness discomfort levels. Loudness growth curves are parallel to those found in normal hearing. Suprathreshold measures such as difference limen for frequency modulated pure tones, gap detection, and particularly speech perception in noise are within the normal range. Distortion otoacoustic emissions are present at the higher stimulus level. These results are similar to those previously obtained from a Dutch DFNA13 family with mid- frequency sensorineural hearing impairment. It seems that a defect in the tectorial membrane results primarily in an attenuation of sound, whereas suprathreshold measures, such as otoacoustic emissions and speech perception in noise, are preserved rather well. The main effect of the defects is a shift in the operation point of the outer hair cells with near intact functioning at high levels. As most test results reflect those found in middle-ear conductive loss in both families the sensorineural hearing impairment may be characterized as a cochlear conductive hearing impairment. Chapter 2.3

Introduction Since 1992, more than 100 types of sensorineural, nonsyndromic hearing impairment have been identified.1 The different chromosomal loci for non- syndromic types of hearing impairment have been designated DFN (DeaFNess) and are numbered in chronological order of discovery. Autosomal dominant types are referred to as DFNA, autosomal recessive types as DFNB, and X-linked types as DFN. These types of hearing impairment can be characterised by age of onset, presence and degree of progression, severity of hearing impairment, and audiometric configuration.2 One of these configurations is the U-shaped or “cookie-bite” audiometric configuration of midfrequency hearing impairment that can be found in DFNA8/12 (TECTA) and DFNA13 (COL11A2).1

The first descriptions of autosomal dominant midfrequency hearing impairment relate to an Austrian (DFNA8) and a Belgian (DFNA12) family.3,4 The original proposed linkage to chromosome 15q in the Austrian family was later on withdrawn and relocated to chromosome 11q22-24.3-5 When the mutations proved to be in the same gene (TECTA), which encodes alpha-tectorin, the locus was redesignated DFNA8/12.6 Alpha-Tectorin is an important noncollagenous component of the tectorial membrane in the cochlea and its level of expression is high during tectorial membrane morphogenesis, between the 12th and 20th week of embryonic development, after which it decreases dramatically.7 The tectorial membrane consists of an extracellular matrix overlying the organ of Corti that contacts the outer hair cells and plays an important role in intracochlear sound transmission by ensuring optimal cochlear feedback.8 Research on transgenic mice showed that the tectorial membrane facilitates the motion of the basilar membrane to optimally drive the inner hair cells.9 The alpha-tectorin protein comprises three distinct modules: the entactin G1 domain, the zonadhesin (ZA) domain, and the zona pellucida (ZP) domain.10 Mutations affecting the ZP domain are significantly associated with midfrequency hearing impairment, whereas mutations in the ZA domain are significantly associated with high frequency hearing impairment.11 Furthermore, mutations in either domain causing substitution of cysteine residues are significantly associated with progressive hearing impairment11.

Legan et al. studied mice with a mutation in the ZP domain of TECTA.9 In these mice with a heterozygous Y1870C mutation (TectaY1870C/+), the tectorial membrane’s matrix structure is disrupted and its adhesion zone is reduced in thickness.9 These

67 Audiological evaluation of DFNA8/12 (TECTA) changes do not seriously influence the tectorial membrane’s role in ensuring optimal cochlear feedback; however, neural tuning is broadened and a decrease in sensitivity is observed at the tip of the neural tuning curve.9

In 2003, De Leenheer et al. studied the audiological characteristics of affected members of a Dutch DFNA13 family.12 DFNA13 is another type of autosomal dominant midfrequency hearing impairment, which is caused by a mutation of COL11A2 on chromosome 6p that also results in alterations of the tectorial membrane.13 In this DFNA13 family, hearing impairment was nonprogressive.12 They observed that in younger subjects suprathreshold signal analyzing capacities are not compromised by the moderate sensorineural hearing impairment, and concluded that DFNA13 results in a cochlear type of conductive hearing impairment.12

In the present DFNA8/12 (TECTA) family nonprogressive midfrequency hearing impairment is caused by a missense mutation substituting arginine residues (R1890C) in the ZP domain of alpha-tectorin.11 The presumably congenital hearing impairment predominantly affects the mid frequencies (1 and 2 kHz), with thresholds around 40 dB. Speech recognition was almost perfect with phoneme recognition scores higher than 90%.11

Such patients with a proven and specific defect in the tectorial membrane provide a unique opportunity to study the audiometric characteristics of tectorial membrane defects. To further outline the audiometric phenotype of DFNA8/12, an extended set of audiological tests was performed on selected affected members of a Dutch DFNA8/12 (TECTA) family. As the tectorial membrane is essential both in cochlear transduction and amplification tests were selected that focus on suprathreshold chacteristics in the time and frequency domain. Outer hair cell functionality was tested separately with distortion product otoacoustic emissions (DPOAE). The results of these tests will be compared to those previously obtained from members of a Dutch DFNA13 (COL11A2) family.12

Methods and material Five adult members of the Dutch DFNA8/12 (TECTA) family, all with a confirmed TECTA gene mutation, were contacted and agreed to participate in this study.11 The gene expression was not significantly different for male and female family members. Subjects participating in this study are indicated as A, B, C, D and E in the

68 Chapter 2.3 pedigree shown in Figure 1. Subjects A to D showed hearing impairment typical for the disorder; subject E only has a relatively mild degree of hearing impairment, but was nevertheless included. Measurements were performed on the ear with the greatest overall impairment.

A B C

D E

Figure 1. Pedigree of the Dutch DFNA8/12 (TECTA) family. Subjects participating in this study are indicated as (A), (B), (C), (D) and (E).11

At the beginning of the experiment pure-tone thresholds were measured with standard audiometric procedures and equipment (Interacoustics AC-40 audiometer). In addition, loudness discomfort levels were measured to provide safe upper limits of stimulus levels when measuring acoustic reflexes. Speech perception was measured in quiet with standard monosyllabic Dutch word lists.14 Each list consists of 11 CVC syllables and scores are based on correct repetition of phonemes. Ipsilateral acoustic reflexes were measured at the pressure providing maximum compliance in the tympanogram (Madsen Zodiac 901 tympanometer).

DPOAE were measured with ILO 292 equipment. The stimuli had a frequency ratio of 1.2, and stimulus levels of 75/60 and 80/70 dB SPL were used. At these levels, artifacts due to nonlinearities in the measurement setup fell below the noise floor. Loudness scaling was performed with a 7-point categorical scale at 500 Hz and 2000 Hz.15 Gap detection was measured with gated white noise and with 500-Hz and 2- kHz noise. Digital filtering of the temporal pattern created the frequency-specific stimuli.

69 Audiological evaluation of DFNA8/12 (TECTA)

Frequency discrimination was measured with frequency-modulated pure tones generated by an audiometer (Interacoustics AC-40). The modulation frequency ranged between 0.1% and 5%. Subjects indicated whether their pitch percept was stable (not modulated) or unstable (modulated). At least three trials aiming at 50% correct performance with a sqrt(2) step size were used. Stimuli were presented at the individual listener’s most comfortable level, i.e. about 40 dB SL.

Speech perception in noise was measured with short, everyday Dutch sentences.16 The speech reception threshold (SRT) was measured with the simple up-down procedure proposed by Plomp and Mimpen.16 SRT will be expressed as a signal-to- noise ratio. All tests were performed in a double-walled sound-treated room. Data will be compared to those for young normal-hearing (NH) listeners.

dB A: 45 yr dB B: 44 yr dB C: 38 yr -10 -10 -10 0 0 0

20 20 20

40 40 40

60 60 60

80 80 80

100 100 100

120 120 120 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz

dB D: 27 yr dB E: 24 yr -10 -10 0 0

20 20

40 40

60 60

80 80

100 100

120 120 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz

Figure 2. Individual hearing thresholds and loudness discomfort levels for subjects (A-E). Air conduction thresholds are indicated with open squares (right ear) and triangles (left ear), and loudness discomfort levels are indicated with black squares (right ear) and triangles (left ear).

70 Chapter 2.3

Results Figure 2 shows pure-tone thresholds and loudness discomfort levels on an individual basis. No substantial air-bone gaps were observed. Subjects are ordered according to their age. All subjects exhibited a hearing impairment between 1 and 4 kHz ranging from about 30 to 60 dB. In all subjects loudness discomfort levels appeared higher than normal.17 Acoustic reflex thresholds obtained with ipsilateral stimulation are shown in Table 1. Most reflex thresholds are elevated compared to the value of 85 dB HL for normal hearing.18

Table 1. Acoustic reflex thresholds in dB HL for ipsilateral stimulation (worst ear) with pure- tone stimuli of 0.5, 1, 2, and 4 kHz.

Subject 0.5 kHz 1 kHz 2 kHz 4 kHz A 100 100 105 >100 B 80 90 90 100 C 85 95 95 100 D 85 95 95 95 E 90 95 95 100

The occurrence of DPOAE is shown in Table 2. In subject A no emissions were measured. At stimulus levels of 80/70 dB SPL emissions were found in the other four subjects; at the lower stimulus level of 75/60 dB SPL, some emissions occurred in subjects B-D. Interestingly, in subject E, who has the most favourable pure-tone thresholds, no emissions occurred at the lowest stimulus level.

Table 2. Distortion product emission (DPOAE) data with 75/60 and 80/70 dB SPL stimuli, measured with ILO-92 equipment (worst ear). Emissions were tested at 1.0, 1.4, 2.0, 2.8, 4.0, and 6.0 kHz. The frequency range at which emissions occur is indicated for each subject.

Subject DPOAE (75/60) DPOAE (80/70) A n.a. n.a. B 4.0 kHz 2.0-6.0 kHz C 1.0, 2.8, 4.0 kHz 1.0-6.0 kHz D 1.4, 4.0 kHz 1.0-6.0 kHz E n.a. 1.0-6.0 kHz

71 Audiological evaluation of DFNA8/12 (TECTA)

Figure 3. Loudness growth functions obtained from the worst ear with 7-point categorical scaling (modified Würzburger Hörfeld Skalierung) for 500-Hz and 2-kHz stimuli. Open circles represent scores for subjects (A-D). Scores for subject (E) are represented by black dots.

Figure 3 shows loudness growth functions for 500-Hz and 2-kHz pure-tone stimuli, obtained with a 7-point categorical scale.15 With the exception of the data for subject E, loudness growth curves are more or less parallel to those for NH subjects,

72 Chapter 2.3 with elevated loudness discomfort levels between 100 and 120 dB HL. Loudness judgments of subject E are within normal limits.

Gap detection was measured with 500-Hz and 2-kHz octave-bands of white noise, and with unfiltered white noise. Results are shown in Figure 4, together with average data for seven naive NH listeners. The measurement for subjects A-D are close to those of NH subjects, with a possible exception for subject D only at 500 Hz. Subject E showed poor results at 500 Hz in this test.

Gap Detection

12

500 Hz 10 2 kHz

8 White Noise

6

4 Gap Detection (ms) Gap

2

0 A B C D E NH Subject

Figure 4. Gap detection with white noise and with 500-Hz and 2-kHz octave bands of noise. Data are shown for subjects (A-E) (worst ear) and for normal-hearing listeners (NH).

Frequency discrimination as expressed in the Difference Limen for frequency (DLf) was measured at 500 Hz and 2 kHz. Data are shown in Figure 5, together with data for normal hearing. The data of subjects A-D, except for the 2-kHz data in subject A, are close to those of the normal subjects. Again, subject E performed relatively poorly on this test.

73 Audiological evaluation of DFNA8/12 (TECTA)

Difference Limen for Frequency

3 500 Hz 2,5 2 kHz

2

1,5 DLf (%)

1

0,5

0 A B C D E NH

Subject

Figure 5. Difference Limen for frequency measured with FM-modulated 500-Hz and 2-kHz stimuli. Data are shown for subjects (A-E) (worst ear) and for normal-hearing (NH) listeners.

All subjects, with the exception of subject A, had a maximum score of 100% for speech perception in quiet (data not shown). Figure 6 shows SRT in noise for sentences. The SRTs are expressed as a signal-to-noise ratio. The data for subjects A- D are within the range of NH subjects (normal limit: -3 dB).16 Alternatively, when expressing the data in Plomp’s D (distortion) term values of +2.4, +0.3, +0.9, +1.1 dB are found for subjects A-D, respectively, and +5.1 dB for subject E. Once more, the data for subject E are outside the normal range.

Discussion Subjects A to D show an elevation of pure-tone thresholds, acoustic reflex thresholds, and loudness discomfort levels. Loudness growth curves are parallel to those found in normal hearing. Suprathreshold measures such as DLf, gap detection, and particularly speech perception in noise are within the normal range. Distortion otoacoustic emissions are present at the higher stimulus level. These findings suggest normal cochlear function at higher stimulus levels. This contrasts strongly

74 Chapter 2.3 to results normally found in sensorineural hearing impairment resulting from loss of outer and/or inner hair cells (e.g., presbyacusis, noise induced hearing loss).19.

Speech Perception in Noise

Subject A B C D E NH 0

-1

-2

-3

-4 SRT (dB S/N ratio)

-5

-6

Figure 6. Speech Reception Thresholds in noise for sentences. Data are shown for subjects (A-E) (worst ear) and for normal-hearing (NH) listeners.

We have no explanation for the relatively poor results found in subject E, whereas his pure-tone thresholds were the most favorable (Figure 2). The hearing characteristics of subject E resemble more closely sensorineural impairment due to hair cell loss. Subject E is thus clearly breaking the trend set by subjects A to D. In view of the unexpected degree of variation in findings, one might wish to extend the number of reliable observations. Unfortunately, this is not an option for the present family. However, the affected family members that were as yet too young to include in this study may be examined later. Alternatively, additional families with a similar phenotype may be recruited for new studies.

It emerges from the present study that a defect in the tectorial membrane reduces sound transduction, resulting in sound attenuation. Suprathreshold measures, such as otoacoustic emissions and speech perception in noise seem to be preserved rather well. The preservation of stimulus fine structure at higher levels is also found in conductive middle ear hearing impairment. This supports the conclusion of De

75 Audiological evaluation of DFNA8/12 (TECTA)

Leenheer et al. that a conductive cochlear type of hearing impairment occurs in tectorial membrane dysfunction.12

The changes observed in the TectaY1870C/+ mouse do not seriously influence the tectorial membrane’s role in ensuring optimal cochlear feedback; however, neural thresholds were elevated and neural tuning curves were broadened.9 Except for the broadening of the neural tuning curves, this TectaY1870C/+ mouse model closely represents the findings in our family. In humans, however, TECTA mostly affects threshold sensitivity with little suprathreshold consequences.

The test results of our subjects are quite similar to the results in subjects with DFNA13 (COL11A2).12 Mice with a targeted disruption of COL11A2 exhibit a loss of organization of the collagen fibrils present in the tectorial membrane, causing pathological alteration in structure of the tectorial membrane that reduces efficacy of the outer hair cells, resulting in congenital hearing impairment that varies in degree from mild to moderately severe.13 More data are needed on the suprathreshold hearing characteristics of COL11A2 mice.

In recent years, mouse models provided a lot of insight in the function of the tectorial membrane and the basis of these types of autosomal dominant mid- frequency hearing impairment.9-10,13 However, the exact mechanism of mutations altering protein interactions and thus influencing the mechanotransductional properties of the tectorial membrane remains uncertain.

In conclusion, the cochlear loss observed in selected members of a DFNA8/12 family acts purely as an attenuation, a result of a reduced efficiency in the coupling of the outer hair cells and the tectorial membrane. These findings are in line with previous observations in DFNA13.12 Although the underlying mutations have different effects on the structure of the tectorial membrane, the common effect of either disorder may be characterised as a cochlear conductive hearing impairment. More precise consequences of TECTA can only be stated with more certainty after studying affected human temporal bones.

76 Chapter 2.3

References 1. Van Camp G and Smith RJH. Hereditary Hearing Loss Homepage. http://webhost.ua.ac.be/hhh/ Accessed June 2006. 2. Huygen PLM, Pauw RJ, Cremers CWRJ. Audiometric profiles associated with genetic non- syndromic hearing impairment, a review and phenotype analysis. Audiol Med 2006. 3. Kirschhofer K, Kenyon JB, Hoover DM et al. Autosomal-dominant, prelingual, nonprogressive sensorineural hearing loss: localization of the gene (DFNA8) to chromosome 11q by linkage in an Austrian family. Cytogenet Cell Genet 1998;82:126-130. 4. Verhoeven K, Van Camp G, Govaerts PJ et al. A gene for autosomal dominant nonsyndromic hearing loss (DFNA12) maps to chromosome 11q22-24. Am J Hum Genet 1997;60:1168-1173. 5. Verhoeven K, Van Laer L, Kirschhofer K et al. Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat Genet 1998;19:60-62. 6. Mustapha M, Chardenoux S, Nieder A et al. A sensorineural progressive autosomal recessive form of isolated deafness, DFNB13, maps to chromosome 7q34-q36. Eur J Hum Genet 1998;6: 245-250. 7. Pfister MHF, Thiele H, Van Camp G et al. A genotype-phenotype correlation with gender-effect for hearing impairment caused by TECTA mutations. Cell Physiol Biochem 2004;14:369-376. 8. Legan PK, Lukashkina VA, Goodyear RJ et al. A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 2000;28:273-285. 9. Legan PK, Lukashkina VA, Goodyear RJ et al. A deafness mutation isolates a second role for the tectorial membrane in hearing. Nat Neurosci 2005;8:1035-1042. 10. Legan PK, Rau A, Keen JN et al. The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system. J Biol Chem 1997;272:8791- 8801. 11. Plantinga RF, de Brouwer AP, Huygen PLM et al. A Novel TECTA Mutation in a Dutch DFNA8/12 Family Confirms Genotype-Phenotype Correlation. J Assoc Res Otolaryngol 2006;7:173-181. 12. De Leenheer EMR, Bosman AJ, Kunst HPM et al. Audiological characteristics of some affected members of a Dutch DFNA13/COL11A2 family. Ann Otol Rhinol Laryngol 2004;113:922-929. 13. McGuirt WT, Prasad SD, Griffith AJ et al. Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13). Nat Genet 1999;23:413-419. 14. Bosman AJ and Smoorenburg GF. Intelligibility of Dutch CVC syllables and sentences for listeners with normal hearing and with three types of hearing impairment. Audiology 1995;4:260-284. 15. Moser LM. Das Würzburger Hörfeld - ein Test für prothetische Audiometrie. HNO 1987;35:318- 321. 16. Plomp R and Mimpen AM. Improving the reliability of testing the speech reception threshold for sentences. Audiology 1979;18:43-52. 17. Pascoe DB. Clinical measurement of the auditory dynamic range and their relation to formulas for hearing aid gain. Proc. 13th Danavox symp.:1988. 18. Silman S and Gelfand SA. The relationship between magnitude of hearing loss and acoustic reflex threshold levels. J Speech Hear Disord 1981;46:312-316. 19. Moore BCJ. Perceptual Consequences of Cochlear Damage. 1995.

77

Chapter 3

Usher syndrome type III (USH3)

3.1

Serial audiometry and speech recognition findings in Finnish Usher syndrome type III patients

Rutger F. Plantinga, Leenamaija Kleemola, Patrick L.M. Huygen, Tarja Joensuu, Eeva- Marja Sankila, Ronald J.E. Pennings, Cor W.R.J. Cremers. Audiology and Neuro-Otology 2005; 10: 79-89. Abstract Audiometric features, evaluated by serial pure tone audiometry and speech recognition tests (n=31), were analysed in 59 Finnish Usher syndrome type III patients with Finmajor/Finmajor (n=55) and Finmajor/Finminor (n=4) USH3A mutations. These patients showed a highly variable type and degree of progressive sensorineural hearing impairment: from normal to moderate USH2A- like hearing impairment at young ages to profound or even USH1B-like hearing impairment at more advanced ages. Compound heterozygous patients generally showed a milder phenotype. The highest progression was seen during the first two decades of life, gradually slowing down with further ageing. This type of nonlinear progression may be unique amongst the Usher syndromes. Speech recognition started to deteriorate at highly variable ages. In some patients, it jeopardised normal speech and language development, whereas in others it was still remarkably good at advanced ages. Chapter 3.1

Introduction Usher syndrome, an eponym of the Scottish ophthalmologist Charles Usher (1865- 1942), is an autosomal recessive disorder that is characterised by the combination of sensorineural hearing impairment (SNHI) and progressive retinitis pigmentosa (RP).1 Some Usher syndrome patients also have vestibular dysfunction. The exact prevalence of Usher syndrome is still unclear. Estimates range from 3.5 to 6.2 per 100,000, making it the most common cause of deaf-blindness worldwide.2 In 1977, Davenport and Omenn introduced a clinical classification of Usher syndrome and after several modifications it now comprises three different types (I-III).3

Usher syndrome type I (USH1) is characterised by congenital, profound SNHI, RP and vestibular areflexia. Until now, 7 chromosomal loci have been identified for USH1 (USH1A-G). Usher syndrome type II (USH2) shows a moderate-to-severe congenital highfrequency SNHI, RP and intact vestibular responses. Three loci have been identified for USH2 (USH2A-C). Usher syndrome type III (USH3) generally shows postlingual, progressive SNHI, RP and variable vestibular responses. Only one locus has been identified: USH3. At present, 7 genes (USH1B/MYO7A, USH1C/USH1C, USH1D/CDH23, USH1F/PCDH15, USH1G/SANS, USH2A/USH2A, USH3/USH3A) have been cloned.4 The genetic subtypes USH1B and USH2A account for about 75-80% of all Usher patients worldwide, whereas USH3 is rare.5

In 1989 Karjalainen et al.6 reported a remarkably high prevalence of progressive hearing impairment in Usher syndrome patients in Finland. After a nationwide study, Pakarinen et al.7 concluded that USH3 accounts for 40% of all Finnish Usher patients and therefore is the most common form of Usher syndrome in Finland. This can be understood by taking notion of the Finnish disease heritage being the consequence of the Finnish population history, which is characterised by the presence of national and regional isolates with rapid expansion.8 Some mutations have been enriched all over the country and probably have been imported by early immigrants to Finland.9 The associated Finnish founder mutation identified in nearly all USH3 cases is the Finmajor (c.300 T>G; c.528 T>G according to the new nomenclature) mutation in the USH3A gene on chromosome 3q21-q25 that changes a tyrosine into a premature stop codon (Y100X; now Y176X) and, when homozygous, leads to a truncated protein.10,11 The change in nomenclature is the result of a newfound exon, discovered in 2002 by Adato et al. Another associated mutation is the Finminor (c.131 T>A; now c.359 T>A) mutation resulting in M44K (now M120K).10,11

83 Hearing impairment in USH3

The aim of this study was to perform a detailed audiometric analysis of the phenotype of USH3 patients with a confirmed genotype. In 1995, Pakarinen (now Kleemola) et al. presented part of the results from the nationwide study mentioned above.12 It comprised the clinical manifestations, including audiometric, ophthalmologic and vestibular examination results, of 42 USH3 patients. Unfortunately, this report proved hard to obtain through medical library services in non-Scandinavian countries and remained fairly unknown. Close co-operation resulted in the present report, which comprises a more detailed analysis of the audiometric findings bearing on an extended group of Finnish USH3 patients, whose genotype had been established in the meantime (n=59).

Patients and Methods

Subjects This study initially comprised 63 Finnish USH3 patients, who had been selected because of their apparent progressive SNHI. Blood samples were collected from the patients for linkage and mutation analysis. Linkage and mutation analysis of this large sample of USH3 patients revealed the USH3A gene, which is described in separate papers.10,11 Fifty-nine of them appeared to have homozygous Finmajor mutations and 4 patients had compound heterozygous Finmajor/Finminor mutations in the USH3A gene. After the exclusion of the patients of whom only one audiogram could be obtained, 59 patients were available for longitudinal analysis of SNHI.

Clinical examinations and audiometry Medical history taking and ophthalmological examinations were performed on all affected individuals.7,12 The latter included external eye examination, corrected visual acuity measurements, Goldmann perimetry, slit-lamp microscopy, ophthalmoscopy and electroretinography. The results of these examinations confirmed a diagnosis of RP in all affected individuals.12 The patients were clinically diagnosed to have USH3 on the basis of medical history and the observed progressive SNHI.

84 Chapter 3.1

Audiometric examination consisted of pure tone audiometry according to the norms of the International Organization for Standardization.13 At ages 2-5 years, the first examinations comprised behavioural (free-field) audiometry.

Speech recognition scores (percent correct recognition) were evaluated in 34 patients (3 with compound heterozygous mutations) using phonetically balanced Finnish word lists.12 For each patient, the binaural mean maximum discrimination score was derived from the two separate performance-intensity plots established for monaural speech presentation.

Statistical analyses Individual linear longitudinal regression analysis (binaural mean air conduction threshold on age) was performed in all patients with sufficient (n=3 or greater) longitudinal auditory threshold measurements covering a suitable follow-up interval. A commercial program (Prism 4, GraphPad, San Diego, CA, USA) was used for plotting and analysing the threshold data. The slope was called annual threshold deterioration (ATD) and expressed in dB/year. Progression (ATD) was designated significant when a significant positive slope (p < 0.025) was found. Systematic significant progression of SNHI was concluded to exist if slopes of a significant proportion of the measured audio frequencies were significantly positive (p < 0.05, according to the appropriate binomial distribution).

Individual longitudinal regression lines were used for constructing individual Age- Related Typical Audiograms (ARTA), i.e. threshold plotted in a compound audiogram format for ages in decade steps.14 It should be emphasised that the ARTA were limited to the frequency range of 0.25-8 kHz for compatibility with previously reported ARTA. The purpose of applying ARTA was twofold: (1) it enabled a simplified presentation of the often large number of serial individual audiograms in a compact audiogram-like format with a clearer view on individual progression and (2) it enabled simple age-corrected intersubject comparison between audiograms, as well as simple comparison between individual ARTA and the overall ARTA (see below), which was used as a reference, to outline intersubject variability in hearing thresholds. For the second application, the individual mean across-frequencies (0.25- 8 kHz) threshold was calculated for a given audiogram obtained at a given age or for all the audiograms pertaining to the fixed ages that constituted the ARTA. This mean threshold was compared to the mean threshold obtained by performing

85 Hearing impairment in USH3 exactly the same calculations using overall ARTA data, either at the appropriate precise age (for the evaluation of a single individual audiogram) or at the fixed ages (in decade steps) involved. Individual ARTA were only derived for methodological reasons and not for the purpose of individual presentation.

Individual longitudinal binaural mean speech recognition scores, as well as single- snapshot scores, were plotted against age and binaural mean PTA1,2,4 kHz (pure tone average at 1, 2 and 4 kHz) level. A trend line was “fitted” where possible in either of these plots in the way described in the Results section. The 90% correct score (X90) was designated onset age for X=age and onset level for X=PTA1,2,4 kHz. The slope was called deterioration rate in the performance-age plot and deterioration gradient in the performance-impairment plot.

The equation of a saturation hyperbola with variable onset age was employed using nonlinear cross-sectional regression analysis to fit the mean thresholds that were derived for various ages to characterise the development of threshold with advancing age (details below):

Y=Bmax (X - Xonset)/(Kd + (X – Xonset)),

where Y is threshold (dB HL), X is age (year), Bmax is the maximum (saturation) threshold (dB HL), Kd (year) is a curvature parameter with the same units as age (the smaller the Kd, the greater the curvature). Details about the hyperbola and the fitting procedure have been previously described.15 In the present modification of the nonlinear regression method, age (X) was substituted by age minus onset age

(Xonset), to the effect that also an estimate of onset age with its 95% confidence interval was obtained.

Results

Pure tone thresholds Serial audiograms (n=2 - 27) were obtained at ages between 2 and 54 years and covered a follow-up interval of 1 - 40 years. Over 400 audiograms were analysed for this study.

86 Chapter 3.1

dB Pat 01 dB Pat 05 dB Pat 09 dB Pat 13 dB Pat 56 dB Pat 17 -10 -10 -10 -10 -10 -10 3,32 6,27 6,31 4,91 13,45 19,17 0 0 0 0 0 0 4,41 5,98 6,5 5,52 14,12 22,15 24,44 20 20 20 20 20 20 5,30 6,02 6,9 5,9 14,59 27,01 40 40 40 40 40 40 7,57 6,6 7,25 6,19 15,02 7,78 60 6,89 60 7,42 60 7,01 60 15,52 60 60 8,71 7,25 8,34 7,62 16,05 80 80 80 80 80 80 13,37 8,82 8,37 17,27 19,63 100 100 10,01 100 8,2 100 19,17 100 100 20,33 120 120 120 13,31 120 20,95 120 120 .125.25.51248 k Hz .125.25.51248 k Hz .125.25 .5 1 2 4 8 kHz .125 .25 .5 1 2 4 8 kHz .125.25 .5 1 2 4 8 kHz .125 .25.51248 kHz

dB Pat 21 dB Pat 25 dB Pat 29 dB Pat 57 dB Pat 58 dB Pat 33 -10 -10 -10 -10 -10 -10 0 6,00 0 7,75 0 8,67 0 15,02 0 22,52 0 11,2 9,00 9,31 13,12 18,14 38,54 23,59 12,16 20 20 20 20 20 20 15,00 11,69 20,0 19,08 38,83 25,57 16,17 40 20,15 40 13,62 40 32,21 40 21,72 40 40,23 27,03 40 21,65 60 20,36 60 16,96 60 34,91 60 27,89 60 27,84 60 28,30

80 22,97 80 18,41 80 80 32,15 80 31,84 80 29,73 23,33 21,03 35,58 35,67 32,36 100 26,13 100 25,71 100 100 100 36,02 100 34,36 120 30,32 120 32,17 120 120 120 37,03 120 37,61 .125.25.51248 kHz .125.25 .5 1 2 4 8 kHz .125.25 .5 1 2 4 8 kHz .125 .25 .5 1 2 4 8 kHz .125 .25.51248 kHz .125 .25.51248 k Hz

dB Pat 59 dB Pat 37 dB Pat 41 13,56 dB Pat 45 8,6 dB Pat 49 dB Pat 53 -10 -10 18,0 -10 -10 11,22 -10 -10 0 20,80 0 0 26,16 0 0 36,18 0 51,63 21,0 13,03 21,22 41,58 26,25 41,30 57,89 20 20 24,0 20 20 13,62 20 20 26,17 42,78 30,49 45,89 60,54 32,0 19,23 40 34,54 40 40 44,00 31,43 40 40 47,47 40 64,10 41,0 24,68 60 34,65 60 60 33,77 60 60 48,89 60 30,08 34,96 34,68 55,47 80 80 80 80 33,65 80 80 36,01 34,98 59,07 100 100 100 36,30 100 42,53 37,67 100 100 41,71 37,76 59,83 38,92 46,98 39,73 120 40,14 120 120 120 120 120 .125.25.51248 kHz .125.25.51248 k Hz .125.25.51248 kHz .125 .25 .5 1 2 4 8 kHz .125.25 .5 1 2 4 8 kHz .125 .25.51248 k Hz

Figure 1. Audiograms obtained from a selection of the 55 Finmajor/Finmajor patients, i.e. patient No. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49 and 53 (unshaded panels) and the 4 Finmajor/Finminor patients, i.e. No. 56-59 (shaded panels). Binaural mean pure tone air conduction thresholds are shown at 0.125-8 kHz. Patients ordered (from top left to bottom right) by age (years) at last visit.

Figure 1 presents a selection of audiograms from patients with the Finmajor/ Finmajor genotype in unshaded panels and the Finmajor/Finminor genotype in shaded panels; the panels are ordered by the patient’s age (indicated in years) at the last visit. In the patients with the Finmajor/Finmajor genotype, the audiogram configuration varied from gently to steeply downsloping. These patients showed a highly variable type and degree of progressive SNHI: from normal to moderate hearing impairment at young ages to profound hearing impairment at more advanced ages. The moment when only residual hearing was left could be reached in the first up to the eighth decade of life. Moderate-to-severe SNHI was already present at the first evaluation in one of the first decades of life or even in early childhood. There were a few notable exceptions: patient 46 still had a close to normal threshold at the age of 23 and patients 10, 21 and 43 only showed a mild threshold elevation at the ages of 12-14, 6 and 13, respectively (data not shown, except for patient 21). The audiograms of the patients that belong to the same

87 Hearing impairment in USH3 family were screened, controlling for age, to see whether their family relationship (data not shown) was associated with any special features. Apart from the findings related to the compound heterozygous patients, no such features were found.

The 4 patients with the Finmajor/Finminor genotype (Figure 1, shaded panels) showed similar audiogram configurations as the Finmajor/Finmajor patients, but a less prominent degree of SNHI relative to age, except for patient 58. This will be detailed in one of the next sections.

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Figure 2. Examples of serial audiometry indicating nonlinear progression in 3 different Finmajor/Finmajor patients. Inset shows audiogram at first and last visit.

Progression of hearing impairment Progression, if present, seemed to be most prominent within the first part of the follow-up interval. Figure 2 shows a selection of 3 patients whose follow-up measurements were particularly suggestive of such a type of nonlinear progression. It would seem that the highest degree of progression tended to occur at ages below 10 years. It seemed impossible to conclude whether SNHI had occurred already congenitally or developed rapidly in early childhood. Regression analysis of the

88 Chapter 3.1 patients covered by Figure 2 including only threshold data obtained at age < 10 years produced ATD values at the various frequencies in the range of 5-17 dB/year in patient 15, 6-16 dB/year in patient 28 and 2-7 dB/year in patient 11.

In 40 out of 51 evaluable cases, a pilot longitudinal analysis over the whole follow-up interval produced individual regression lines showing significant progression at 2-7 out of the 7 audio frequencies (significant). The degree of progression and the thresholds involved varied substantially. Figure 3 illustrates this pilot analysis.

1 kHz

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Figure 3. Illustration of data underlying pilot analysis of thresholds at 1 kHz showing (straight) individual longitudinal regression lines. Individual data points have been omitted for clarity. Included is the same curve as shown in Figure 5, which was derived from the hyperbolic fit that was performed on the basis of the cross-sectional data included in Figure 4.

It includes all the straight regression lines that were fitted to the individual longitudinal threshold data at 1 kHz (data points have been omitted for clarity). Also included is the curve, a saturation hyperbola with variable onset age, which was fitted to the cross-sectional mean thresholds derived in relation to Figures 4 and 5 (Patients and Methods). For each frequency, each individual regression line (shown for 1kHz in Figure 3) could be characterised by an estimated median threshold during follow-up and the ATD (data not shown). This could be related to either the

89 Hearing impairment in USH3 corresponding median age during follow-up or the duration of follow-up (data not shown). The ATD appeared to increase significantly with decreasing median age or duration of follow up. The greatest variability in ATD was found at the younger ages (or the shorter durations of follow up; see Figure 3), with tailing towards ATD values as high as 20 dB/year (analysis and data not shown). Such parameter behaviour, although the parameters pertained to (individual, longitudinal) linear regression analyses (covering smaller parts of the overall age interval), is compatible with overall nonlinear progression, as is clearly illustrated for 1 kHz in Figure 3. Apparently, individual progression (as assessed by linear regression analysis over the shorter individual age intervals) tended to be higher at relatively younger ages (or relatively short follow-up intervals) than was reflected by cross-sectional calculations (data not shown). Progression gradually slowed down at a more advanced age (or at relatively long durations of follow up; see Figure 3).

Age-Related Typical Audiograms Figure 4 includes all individual ARTA data plotted in superposition per fixed age (10, 20, 30, 40, 50 and 60 years) in decade steps (second and third rows of panels) that were used to derive overall ARTA (bottom panels). For each frequency, the across- subjects mean threshold was calculated at each fixed age. The top row panels include first-visit audiograms for two subgroups of patients aged below 10 years. Invoking the data for these younger age groups, it was our intention to extend the ARTA towards younger ages by including mean first-visit audiograms calculated directly from the individual thresholds at the corresponding mean age at first visit. However, we provisionally separated the first-visit audiograms for the patients aged 5 years and below from those for the group of patients aged 5-10 years because the younger children mainly had behavioural audiometry (Patients and Methods). The top left panel of Figure 4 clearly shows that the first-visit thresholds for the youngest children (aged 2.1-4.9 years) were substantially higher than for the children aged 5.5-9 years (top right panel). We therefore decided to exclude the threshold data pertaining to the youngest age group from the calculation of ARTA (Discussion). The bottom panels of Figure 4 show a combination of the mean thresholds obtained for each age-related selection of audiograms or ARTA at fixed ages, resulting in an overall ARTA. There is, however, an additional anomaly involved: at all frequencies, the threshold seemed to decrease over the last two decades. Figure 5 shows this more clearly.

90 Chapter 3.1

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Threshold (dB HL) (dB Threshold 120 60 y 110 .125 .25 .5 1 2 4 8 .125 .25 .5 1 2 4 8 Frequency (kHz)

Figure 4. Top panels: first-visit individual audiograms obtained at the ages of 2.1-4.9 years (mean 3.8 years, left) and 5.5-9 years (mean 7.2 years, right panel). Second and third rows of panels: composite audiogram-like plots collected from individual ARTA covering the fixed ages 10, 20, 30, 40 and 60 years. Bottom panels: across-subjects mean threshold for the separate fixed ages.

91 Hearing impairment in USH3

This anomaly must have been caused by the fact that not each of the mean thresholds calculated for the fixed ages (Figure 4) pertained to the whole group of patients. The individual ARTA for each patient covered only a limited number of fixed ages (in decade steps). The patient thus contributed only to the calculation of the corresponding mean thresholds which, taken together, established the overall ARTA for the whole group of USH3 patients. Thresholds calculated for different fixed ages therefore could relate to different subgroups of patients, the oldest of whom apparently had relatively better thresholds. Obviously, this anomaly could never have occurred in individual longitudinal analyses. We therefore excluded the mean thresholds at 60 years of age from the nonlinear regression analysis that was performed to obtain curves approximating the threshold as it increased with increasing age (Figure 5). The equation of a saturation hyperbola with variable onset age was fitted to the remaining mean threshold data (Patients and Methods). The estimates of onset age did not differ significantly across the frequencies according to one-way analysis of variance (data not shown).

120 ATD 9 dB/year kHz 100 8 4 2 80 1 0.5 60 0.25 0.125 40 Threshold (dB HL) (dB Threshold

0 10 20 30 40 50 60 70 Age (year)

Figure 5. Across-subjects mean thresholds taken from Figure 4 (bottom panels), now plotted against the ages 7.2 and 10-60 years to evaluate progression. Vertical bars indicate 1 SE. The curves were fitted with exclusion of age 60 years, using a nonlinear equation (see text). Oblique arrow indicates an ATD of 9 dB/year.

92 Chapter 3.1

The mean across-frequencies onset age was 5.2 years (range 2.2-6.3), however, onset age did not differ significantly from zero (i.e. age zero was included within the estimated 95% confidence interval) at 4-8 kHz (data not shown). As shown in Figure 5, the fitted curves have their highest slope at age 7.2 years and below. The oblique arrow is a reference ATD (slope) of 9 dB/year. This value was chosen for the occasion because in Figure 6, the maximum vertical distance between the thresholds estimated for (exactly) 7 and 10 years of age is 27 dB.

Figure 6 shows the overall ARTA (continuous lines) derived from the curves in Figure 5 (extrapolated to include the ages of exactly 7 and 60 years). The nonlinear progression covered by the ARTA for USH3 in Figure 6 is reflected by threshold increments that gradually decrease with increasing age. ARTA characterising USH1B and USH2A are included in Figure 6 for the sake of comparison (Discussion).

dB -10 0

10 USH2A 2020 30 40 4050 60 60 7 80 10

100 0-60 USH3 20 Threshold USH1B 30-60 120 .25 .5 1 2 4 8 kHz Frequency

Figure 6. Overall ARTA (continuous lines) for Finmajor/Finmajor patients, derived from the curves shown in Figure 5 (extrapolated to include ages 7 and 60). ARTA for USH2A (thin short dashes) 16 and USH1B (bold long dashes) 17 are shown for comparison. Age (in years) is indicated in Italics. It can be noted that the thresholds of USH3 patients aged 7-20 years may overlap with the thresholds of USH2A patients aged 10-60 years.

93 Hearing impairment in USH3

Patients with Finmajor/Finminor mutations Three of the 4 Finnish patients with this genotype (Figure 1, No. 56-59, shaded panels) showed remarkably less SNHI than patients with homozygous Finmajor mutations (No. 1-55). Their mean threshold (0.25-8 kHz) was derived along with their mean age and compared to the mean threshold predicted for that age using the data on which the overall ARTA for the Finmajor/Finmajor patients were based. These mean thresholds were better by 38-40 dB for patients 57 and 59 (their individual ARTA – data not shown - were fairly similar to those of USH2A patients) and by 56 dB for patient 56 (ARTA even better than in USH2A patients), respectively. However, the ARTA of the remaining Finmajor/Finminor patient (No. 58) were very similar to the present overall USH3 ARTA derived for the homozygous Finmajor genotype, with a mean threshold (88 dB) that was worse by 3 dB compared to the mean threshold that was predicted for USH3 at the corresponding mean age (32.1 years) by the nonlinear regression equations underlying (Figure 5) the overall ARTA data.

Speech recognition scores Speech recognition scores are shown in Figure 7. Serial measurements were available in 23 patients with homozygous Finmajor mutations; they comprised 2 - 7 measurements covering an interval of ages between 1 and 25 years. Single-snapshot measurements were available from 8 patients. The phoneme scores are plotted against the patient’s age (Figure 7A) and the level of SNHI (Figure 7B). Figure 7A shows that speech recognition could begin to deteriorate appreciably (score <90%) in individual cases from virtually any (onset) age above 5 years. Given the variability in pure tone audiometry (Figure 3) this could have been expected. There was also a great variability in deterioration rate: individual data indicate values ranging from close to zero up to about 10% per year. The performance-impairment plot (Figure 7B) showed less variability. A trend line was fitted with a presumed deterioration gradient of 1% per dB hearing loss (HL). The intercept of the trend line was visually fitted to obtain almost equal numbers of patients, no matter whether they had single-snapshot or serial measurements that are represented by data points above or below this line. The onset level (PTA at score 90%) thus found was about 70 dB HL.

94 Chapter 3.1

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Figure 7a-b. Evaluation of speech recognition scores relative to age (A, performance-age plot) and PTA1,2,4 kHz (B, performance-impairment plot) in 31 homozygous Finmajor/Finmajor USH3 patients. Open circles and connecting hairlines: longitudinal measurements (23 patients); filled circle: single-snapshot measurement. Bold line: trend line fixed at slope –

1%/dB (see text); dotted lines: onset level (90% score) at 70 dB PTA1-4 kHz.

Longitudinal speech recognition scores were available in 3 of the 4 patients with compound heterozygous Finmajor/Finminor mutations. The scores were fairly similar to those of the homozygous Finmajor patients in relation to age and level of SNHI (data not shown).

Discussion This study comprises a detailed audiometric analysis of the USH3 phenotype in fully genotyped Finnish patients and thus allows for a more precise comparison to the phenotypes found in patients with other types of Usher syndrome. As previously described, the Finnish USH3 patients showed an impressively wide spectrum of SNHI.12 The present report focuses on the issues of nonlinear progression and variation in degree and onset age of SNHI.

Recapitulation of audiometric findings There is no doubt that the majority of the Finnish USH3 patients with homozygous Finmajor mutations covered by this study had experienced substantial progression in SNHI from early childhood onwards. It is unclear whether they also had any substantial congenital SNHI, especially because there were a few exceptions (Results). The results of the hyperbolic fitting procedure that was used to derive overall ARTA suggest an onset age of about 5 years (Figure 5), but this finding should

95 Hearing impairment in USH3 be regarded with great reservation as it derived from applying an arbitrary nonlinear regression model.

From early childhood onwards, the patients showed a great variability in the level of SNHI and the degree of progression. Part of the variability was clearly related to age in a nonlinear way. The highest progression was found at the youngest ages and gradually slowed down at more advanced ages (Figures 2 and 3). Analysis of speech recognition scores in relation to age disclosed that in a small minority of the patients the deterioration of speech recognition started so early and developed so rapidly that the development of normal speech and language skills could have been jeopardised.

Progression as a distinctive feature Based on the results of this study we suggest that USH3 may be unique amongst the Usher syndromes because of its high degree of (nonlinear) progression. USH3 patients showing more moderate progression than is indicated by the present results may have been excluded from the present sample by the clinical selection procedure that was followed. Even if that would be the case, the statement can be upheld that the high degree of progression shown by many of our USH3 patients is unique amongst the Usher syndromes. If the mean threshold at a given age and the degree of progression in SNHI for the whole population of USH3 patients with the Finmajor/Finmajor phenotype would be lower than our present estimates, it would imply that the degree of variability in threshold and progression within that USH3 population is even greater than already could be pinpointed for the present, no doubt selected, USH3 sample.

Selection bias The thresholds in the group of children aged 2.1-4.9 years (Figure 4, top left panel) were substantially higher than those in the group of children aged 5.5-9 years (Figure 4, top right panel). Most of the data in the youngest age group had been obtained using behavioural audiometry. When dealing with serial audiometry in young children, in whom the first threshold measurements have been obtained with behavioural methods, it is not uncommon to find that the first thresholds appear to be higher than those obtained at a more advanced age with the usual clinical methods. However, inspection of the longitudinal data of these children (not shown) demonstrated that this was not the case here. The children appeared to have

96 Chapter 3.1 relatively high thresholds, including those obtained (later) with conventional clinical audiometry. Although this does not exclude the possibility of an upward bias in the behavioural threshold data obtained at the youngest ages, it does suggest the possibility of an overall upward selection bias. Given the procedure of selecting the patients with apparent progression in SNHI prior to mutation analysis, a selection bias favouring high thresholds is bound to develop especially in the lower age classes if that is where the highest degree of progression and variability tend to occur. The latter features were indeed indicated by the results of a pilot analysis that employed simple linear regression analysis (Figure 3) and for each individual related the estimated median threshold and ATD to the median age during follow up or the duration of follow up (Results). For that matter, it is possible that our whole patient sample is biased towards high progression or relatively high thresholds resulting from that progression.

Genotype-phenotype correlation in USH3 Some of the analysed patients with the compound heterozygous combination of Finmajor/Finminor mutations showed relatively better hearing than the homozygous Finmajor patients. This can be explained by the consequences of the mutations. The Finmajor nonsense mutation (Y176X) causes a premature stop codon, which leads to a truncated protein in homozygous patients. The Finminor mutation (M120K) is a missense mutation by which methionine is replaced by lysine and thus probably alters the protein less drastically. Speech recognition, however, seemed to be fairly similar in relation to the level of SNHI. The function of the USH3A protein in the cochlea and retina is still unknown. Adato et al.11 suggested that this protein might be involved in cochlear hair cells and spiral ganglion cells. They speculated that proper function could be maintained in early life by functional redundancy and that time dependent loss of such potential redundancy occurs by unknown reasons. We have no idea as to what could explain the high degree of intersubject variability in the extent of hearing impairment in the group of homozygotes. It was even present within the same sibships (data not shown). The 4 compound heterozygotes showed a similar degree of variability as the homozygotes.

97 Hearing impairment in USH3

Comparison of auditory features of Finnish USH3 patients to those reported for Dutch USH1B and USH2A patients Three previous studies comprised cross-sectional analysis of SNHI in USH2A patients.16-18 The patients studied by van Aarem et al. and Wagenaar et al. were all linked to the USH2A locus. Several of these patients were also included in the study by Pennings et al.16, which only included patients in whom at least one mutation was found in USH2A. SNHI in their USH2A patients was generally less severe and less variable than was found in the present Finnish USH3 patients. The ARTA included in Figure 6 illustrate that USH3 and USH2A patients may show fairly similar progression at least at some ages or age intervals, as is indicated by the threshold increments associated with decade steps in age. Pennings et al.16 found a pooled ATD (across frequencies) of 0.5 dB/year. Figures 1-6 show that substantially higher progression tended to occur in many of the Finnish USH3 patients during the first decades of their life. This type of nonlinear progression displayed by USH3 may be unique amongst Usher syndromes.

As previously suggested18, some of the Finnish USH3 patients with relatively good hearing seemed to show a fair similarity in hearing thresholds to USH2A patients, whereas some of the USH3 patients with relatively poor hearing seemed to have audiograms (Figure 1) that looked fairly similar to those of USH1B patients.17,19 Indeed, the present analyses and comparisons demonstrate that a considerable degree of overlap in audiometric phenotype is possible between USH3 and either USH2A or USH1B.19. This is clearly suggested by the wide spectrum of variability in the audiograms shown in Figure 1 and is illustrated by the ARTA in Figure 6. Obviously, the pertinent difference between USH3 patients with an USH1B-like audiogram and USH1B patients is that in the latter this type of audiogram is congenital and stationary, whereas in the former it represents the final stage of progressive deterioration. A related difference between these two patient groups therefore is that a number of the USH3 patients have normal speech.

The importance of genotyping The degree of overlap in the type of audiogram that is possible between USH1B, USH2A and USH3 is a matter of special concern for the clinical diagnosis of Usher syndrome. Given the high degree of variability in threshold features, phenotyping might best be accomplished by collecting sufficient individual longitudinal data,

98 Chapter 3.1 covering an appropriate age range. A remarkable progressive hearing loss is from a clinical viewpoint most likely compatible with USH3, but even when such data are available it is still needed to base the genetic diagnosis entirely on the genotype obtained through mutation analysis.

Audio-vestibular classification of Usher syndromes Otterstedde et al.20 have suggested a new clinical classification of Usher syndromes that distinguishes two subtypes: “vcUSH1”, a vestibulo-cochlear form and “cUSH1”, a cochlear form of Usher syndrome type I. Their suggestion was based on the finding of patients with USH1B-like audiograms and normal vestibular function. In the Finnish study mentioned earlier, vestibular testing was performed on 17 USH3 patients. Vestibular function was normal in 9 patients (52%) and decreased in 8 patients (48%).12 In the light of these findings, the suggestion seems justified that patients with “cUSH1” may turn out to be USH3 patients. In our opinion, knowing the genotype of the patients involved, as far as molecular genetic analysis goes anyway, is a necessary prerequisite when (re)considering clinical classifications based on phenotype features. We suggest that the current audio-vestibular classification of Usher syndromes can be maintained: however, it should clearly be understood that USH3 patients may have intact vestibular responses and that their distinctive audio-vestibular feature is the typical nonlinear progression in SNHI.

Comparison of speech recognition features between Finnish USH3 and Dutch USH2A patients The USH2A patients studied by Pennings et al.16 showed much more uniformity in speech recognition scores related to age and the level of SNHI than the present USH3 patients. The onset age of deterioration of speech recognition in these USH2A patients could be pinpointed at about 40 years; the onset level was almost equal to the present value of 70 dB in USH3 patients. The deterioration gradient in the USH2A patients was 0.6%/dB, with a 95% confidence interval of 0.4-0.8%.16 Although we were unable to obtain an estimate of the present deterioration gradient by regression because of the presence of many replications (i.e. individual longitudinal data) among the scores, we feel that the performance-impairment plots, including the estimated onset level and deterioration gradient, were fairly similar in USH3 and USH2A patients. For a detailed comparison of these data with previously published data, we refer to Pennings et al.16

99 Hearing impairment in USH3

Conclusions The group of Finnish USH3 patients demonstrated an impressively wide spectrum of progressive SNHI, with hearing thresholds that ranged from almost normal to thresholds that are similar to those found in USH2A or even in USH1B patients. It is uncertain as to whether part of the SNHI is already present at birth or develops from early childhood onwards. Anyway, progression was most impressive during the first and second decades of life and thereafter gradually declined in most patients (Figures 2 and 3). Speech recognition started to deteriorate at highly variable ages. Early deterioration of speech recognition may have jeopardised normal speech and language development in some of the patients, whereas others still had remarkably high recognition scores at a more advanced age despite considerably elevated pure tone thresholds. Because of the high degree of variability in threshold features, it seems preferable to base the diagnosis on the genotype obtained through mutation analysis.

References 1. Usher CH. On the inheritance of retinitis pigmentosa, with notes of cases. R Lond Ophthalmol Hosp Rep 1916;19:130-236. 2. Spandau UHM, Rohrschneider K. Prevalence and geographical distribution of Usher syndrome in Germany. Graefes Arch Clin Exp Ophthalmol 2002;240:495-8. 3. Davenport SLH, Omenn GS. The heterogeneity of Usher syndrome. In: Littlefield JW, Ebbing FJG, Henderson JW (eds). Fifth International Conference on Birth Defects. Amsterdam, Excerpta Medica 1977:87-8. (abstract) 4. Van Camp G, Smith RJH. Hereditary Hearing Loss Homepage (HHH). Available from: http://www.uia.ac.be/dnalab/hhh/. Accessed June 2003 5. Pennings RJE, Wagenaar M, Aarem A van, Huygen PLM, Kimberling WJ, Cremers CWRJ. Hearing impairment in Usher’s syndrome. In Cremers CWRJ, Smith RJH (eds), Genetic hearing impairment. Its clinical presentations. Adv Otorhinolaryngol 2002;61:184-91. 6. Karjalainen S, Pakarinen L, Terasvirta M, Kaariainen H, Vartiainen E. Progressive hearing loss in Usher's syndrome. Ann Otol Rhinol Laryngol 1989;98:863-6. 7. Pakarinen L, Karjalainen S, Simola KOJ, Laippala P, Kaitalo H. Usher’s syndrome type 3 in Finland. Laryngoscope 1995;105:613-7. 8. Norio R, Nevanlinna HR, Perheentupa J. Hereditary diseases in Finland; rare flora in rare soul. Ann Clin Res 1973;5:109-41. 9. Peltonen L, Jalanko A, Varilo T. Molecular genetics of the Finnish disease heritage. Hum Mol Genet 1999;8:1913-23. 10. Joensuu T, Hämäläinen R, Yuan B, Johnson C, Tegelberg S, Gasparini P, Zelante L, Pirvola U, Pakarinen L, Lehesjoki A-E, Chapelle A de la, Sankila E-M. Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3. Am J Hum Genet 2001;69:673-84.

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11. Adato A, Vreugde S, Joensuu T, Avidan N, Hamalainen R, Belenkiy O, Olender T, Bonne-Tamir B, Ben-Asher E, Espinos C, Millian JM, Lehesjoki AE, Flannery JG, Avraham KB, Pietrokovski S, Sankilla EM, Beckman JS, Lancet D. USH3A transcripts encode clarin-1, a four-transmembrane- domain protein with a possible role in sensory synapses. Eur J Hum Genet 2002;10:339-50. 12. Pakarinen L, Sankila E-M, Tuppurainen K, Karjalainen S, Kääriänen H. Usher syndrome type III (USH3): the clinical manifestations in 42 patients. Scand J Logoped Phoniatr 1995;20:141-50. 13. International Organization for Standardization. ISO 8253-1: Acoustics: Audiometric Test Methods, I: Basic Pure Tone Air and Bone Conduction Threshold Audiometry. Geneva, Switzerland, International Organization for Standardization, 1989. 14. Huygen PLM, Pennings RJE, Cremers CWRJ. Characterizing and distinguishing progressive phenotypes in nonsyndromic autosomal dominant hearing impairment. Audiol Med 2003;1:37- 46. 15. Cremers CWRJ, Admiraal RJC, Huygen PLM, Bolder C, Everett LA, Joosten FBM, Green ED, Van Camp G, Otten BJ. Progressive hearing loss, hypoplasia of the cochlea and widened vestibule aqueducts are very common features in Pendred’s syndrome. Int J Pediatr Otorhinolaryngol 1998;45:113-23. 16. Pennings RJE, Huygen PLM, Weston MD, Aarem A van, Wagenaar M, Kimberling WJ, Cremers CWRJ. Pure tone hearing thresholds and speech recognition scores in Dutch patients carrying mutations in the USH2A gene. Otol Neurotol 2003;24:58-63. 17. Wagenaar M, Aarem A van, Huygen PLM, Pieke-Dahl S, Kimberling WJ, Cremers CWRJ. Hearing impairment related to age in Usher syndrome type 1B and 2A. Arch Otolaryngol Head Neck Surg 1999;125:441-5. 18. Aarem A van, Pinckers AJLG, Kimberling WJ, Huygen PLM, Bleeker-Wagemakers EM, Cremers CWRJ. Stable and progressive hearing loss in type 2A Usher’s syndrome. Ann Otol Rhinol Laryngol 1996;105:962-7. 19. Pennings RJE, Fields RR, Huygen PLM, Deutman AF, Kimberling WJ, Cremers CWRJ. Usher syndrome type III can mimic other types of Usher syndrome. Ann Otol Rhinol Laryngol 2003;112:525-30. 20. Otterstedde CR, Spandau U, Blankenagel A, Kimberling WJ, Reisser C. A new clinical classification for Usher’s syndrome based on a new subtype of Usher’s syndrome type I. Laryngoscope 2001;111:84-6.

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3.2

Visual impairment in Finnish Usher syndrome type III

Rutger F. Plantinga, Ronald J.E. Pennings, Patrick L.M. Huygen, Eeva-Marja Sankila, Kaija Tuppurainen, Leenamaija Kleemola, Cor W.R.J. Cremers, August F. Deutman. Acta Ophthalmologica Scandinavica 2006;84:36-41. Abstract Purpose: To Evaluate of visual impairment in Finnish Usher syndrome type 3 (USH3) and compare this with Usher syndrome types IB (USH1B) and 2A (USH2A). Methods: We carried out a retrospective study of 28 Finnish USH3 patients, 24 Dutch USH2A patients and 17 Dutch USH1B. Cross-sectional regression analyses of the functional acuity score (FAS), functional field score (FFS*) and functional vision score (FVS*) related to age were performed for all patients. The FFS* and FVS* were calculated using the isopter V-4 test target instead of the usual III-4 target. Statistical tests relating to regression lines and Student’s t-test were used to compare between USH3 patients and the other genetic subtypes of Usher syndrome. Results: Cross-sectional analyses revealed significant deterioration in the FAS (1.3 % per year), FFS* (1.4 % per year) and FVS* (1.8 % per year) with advancing age in the USH3 patient group. At a given age the USH3 patients showed significantly poorer visual field function than the USH2A patients. Conclusions: The rate of deterioration in visual function in Finnish USH3 patients was fairly similar to that in Dutch USH1B or USH2A patients. At a given age, visual field impairment in USH3 patients was similar to that in USH1B patients but poorer than in USH2A patients. Chapter 3.2

Introduction Usher syndrome, an eponym of the Scottish ophthalmologist Charles Usher (1865- 1942), is an autosomal recessive disorder that is characterised by the combination of progressive retinitis pigmentosa (RP) and sensorineural hearing impairment (SNHI).1 Usher syndrome is estimated to occur in 3.5 to 6.2 in 100,000, making it the most common cause of deaf-blindness worldwide2 Usher syndrome is divided into three clinical types (I-III) on the basis of audiovestibular features and 13 corresponding genotypes.3

Usher syndrome type I (USH1A-G) is characterised by congenital, profound SNHI, RP and vestibular areflexia. Usher syndrome type II (USH2A-C) shows moderate to severe congenital high frequency SNHI, RP and intact vestibular responses. Usher syndrome type III (USH3) shows a highly variable type and degree of progressive SNHI, RP and variable vestibular responses.4 The genetic subtypes USH1B and USH2A account for about 75-80% of all Usher patients worldwide, whereas Usher syndrome type III is rare.5 However, Usher syndrome type III is common in the Scandinavian countries and especially in Finland. Pakarinen et al.6 concluded that USH3 accounts for 40% of all Finnish Usher syndrome patients.

A Finnish founder mutation is associated with the high prevalence of USH3 in the Finnish Usher syndrome population.7 This mutation in the USH3A gene on chromosome 3q21-q25 was designated Finmajor (c.300 T>G; c.528 T>G according to the new nomenclature) and leads to a premature stop codon for a tyrosine at amino acid position 176 (Y176X, previously Y100X).8-9 It has currently been identified in over 44 Finnish families as the cause of USH3 (unpublished results). The change in nomenclature is the result of a newly identified exon that was discovered by Adato et al. Another associated mutation is the Finminor (c.131 T>A; now c.359 T>A) mutation resulting in M120K (previously M44K).8,9 Retinitis pigmentosa leads to impaired dark adaptation, progressive visual field constriction and reduction in visual acuity (VA) in all types of Usher syndrome and may even result in blindness. Patients with USH3 have been reported to have hypermetropia with astigmatism as opposed to hypermetropia without astigmatism in USH1 and myopic refractive errors in USH2.10 Overall, 50% of patients with Usher syndrome at some point develop posterior subcapsular cataract that may severely hamper vision.11

105 Visual impairment in USH3

In 1996, Pakarinen et al. presented a report on the ophtalmological course of Usher syndrome type III patients. However, it only included VA data for analysis and comparison between the different clinical types of Usher syndrome.10 At that time, the different genotypes in Usher syndrome had scarcely been identified. The VA and visual field measurements of genetically confirmed Finnish USH3 patients were re- analysed in the present study and compared to similar analyses of Dutch USH1B and USH2A patients, identified by mutations in the MYO7A and USH2A genes, respectively.12

Patients and methods

Subjects This study included 28 patients (25 Finmajor-Finmajor and three Finmajor-Finminor) with a clinically and genetically confirmed diagnosis of Usher syndrome type III.8 Routine ophthalmological examination included external eye examination, corrected VA measurements, testing of refraction, Goldmann perimetry, biomicroscopy and ophthalmoscopy. We included only those patients whose VA scores and visual fields had been evaluated at least once. Data and patients were collected retrospectively after a previous nationwide epidemiological study of Usher syndrome type III in Finland conducted during 1989 – 1992. Medical records were obtained, in accordance with local ethics committees and in co-operation with the Finnish Ministry of Health, to include as many VA and visual field measurements as possible. Data on vision field function were accepted without a priori criteria, provided that functions had been reliably measured and data sufficiently documented. The patients enrolled in this study were not selected according VA or visual field findings.

Evaluation of visual acuity and visual field deterioration Visual acuity was measured by using standard Snellen E-charts. Best corrected measurements of both eyes were used for further evaluation. The VA measurement for each eye was converted into a visual acuity score (VAS) according to Weber- Fechner’s law. The functional acuity score (FAS) was determined by the equation FAS 13 = (3 x VASboth eyes + VASleft eye + VASright eye)/5.

106 Chapter 3.2

Visual field size was evaluated by Goldmann perimetry in both eyes. The visual field score (VFS), as defined by the American Medical Association in their 5th edition of the Guides for the Evaluation of Permanent Impairment13, is related to the III-4 target. Unfortunately, the available Finnish USH3 visual field data mainly involved the V-4 target and the III-4 target had hardly been measured. For this reason, visual field scores were quantified by plotting the V-4 isopter instead of the III-4 isopter; the derived (V-4) scores are marked with an asterisk to designate the different isopter. The corresponding visual field score (VFS*) for each eye separately and both eyes combined were obtained by drawing 10 meridians in the visual field examination form; two in each upper quadrant (at 25, 65, 115 and 155 degrees) and three in each lower quadrant (at 195, 225, 255, 285, 315 and 345 degrees). These VFS*s were converted to a functional field score (FFS*) using a similar equation as for the conversion of the VAS. Finally, the functional vision score (FVS*) was determined by the FAS and the FFS* based on the equation FVS* = (FFS* x FAS)/100.13

To allow for a correct comparison of functional vision scores between the present Finnish USH3 patient group and the previously published Dutch USH1B and USH2A patient groups12, the field scores for the V-4 target (FFS* and FVS*) were derived in the latter two patient groups as well. Special attention was paid to the quantitative relationship between the parameters based on the III-4 target (FFS and FVS, precisely as recommended by the American Medical Association) and the corresponding newly defined parameters (FFS* and FVS*, respectively) based on the V-4 target. In total, 17 USH1B and 24 USH2A patients had V-4 Goldmann perimetry examinations and thus were included in the present analyses.

Statistical analysis Regression analysis was used to analyse cross-sectional functional visual score data (FAS, FFS* or FVS*) for the Finnish USH3 patients in relation to patient age. Deterioration was concluded to be significant if the correlation coefficient was negative at a probability level of < 0.025. The regression data obtained for the functional visual scores (FAS, FFS* and FVS*) for the Finnish USH3 patients were compared to the corresponding scores bearing on the Dutch USH1B and USH2A patients.12 The regression lines were compared using a procedure (Prism program, version 4, GraphPad, San Diego, California, USA) fairly similar to covariance analysis. Regression lines were concluded to differ significantly if either the F-test pertaining to the comparison between the slopes of these lines

107 Visual impairment in USH3 produced a significant value (p < 0.05), or - when the slopes were not significantly different - if the F-test pertaining to the comparison between the elevations of the lines (i.e. a measure for dispersion comprising grand means of both x and y values for each subset of regression data) produced a significant value. Regression lines were also compared using an optimum age window for the comparison in question. Non-linear regression analysis was used to find the age at which the FVS* was 50% and a 95% confidence interval (CI) was also obtained for this estimate.

(%) FAS (%) FFS*

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40 40

20 20

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Figure 1. FAS, FFS* and FVS* for USH3, FinMajor homozygotes (solid triangles) plotted together with the corresponding data for the Dutch USH1B (dots) and USH2A (open circles) patients. Regression lines (dotted line, USH3; solid line, USH1B; dashed line, USH2A) are included. Bold regression line indicates significant deterioration. Small symbols represent outlying values, which were excluded from regression analyses. Vertical hairlines show age window (22-48 y). FinMajor/FinMinor compound heterozygotes are also plotted (solid squares) but these data were excluded from regression and subsequent analyses.

108 Chapter 3.2

Results

Functional visual scores in USH3 patients compared to those of USH1B and USH2A patients (cross-sectional analysis) Figure 1 shows the FAS, FFS* and FVS* data for USH3 plotted together with those for the Dutch USH1B and USH2A as previously published.12 All scores deteriorated significantly for the USH3 patients. Although many of the FAS data for USH3 patients tended to be lower at more advanced ages than those measured for USH1B or USH2A patients, there was no significant difference found between the regression lines. Comparison of the FFS* or the FVS* data between the patient groups showed a significant difference between the elevations of the regression lines: at a given age, the highest scores belonged to the USH2A patients, whilst the lowest scores belonged to the USH3 patients. The significant difference could be attributed to the USH3 and USH2A groups. Corresponding differences were found between the ages at which the FVS* equalled 50%. These ages were estimated at 28 for USH3 and 39 for USH2A; the estimated 95% CIs did not overlap (Table 1).

Table 1. Slopes (score “% change per year”) for the cross-sectional regression lines relating to the FAS, FFS* and FVS* for USH3, USH1B and USH2A. Pooled slopes are included. The slopes relating to the FFS or FVS (based on test target III-4 and previously published by Pennings et al. 12) for USH1B and USH2A are shown in parentheses for comparison.

FAS FFS* (FFS) FVS* (FVS) Age at FVS* 50% (95% CI)

USH3 -1.3 -1.4 -1.8 28 (24-32)

USH1B -0.6 -1.6 (-1.3) -1.6 (-1.5) 33 (28-38)

USH2A -0.8 -0.9 (-1.0) -1.6 (-1.4) 39 (35-44)

pooled -0.9 -1.3 -1.7

There was no significant difference in slope detected between the three patient groups. Applying the age window (Figure 1) did not introduce essential changes. Table 1 lists the slopes for the cross-sectional regression lines relating to the FAS, FFS* and FVS* for the separate groups USH3, USH1B and USH2A; pooled slopes are included. The slopes relating to the FFS for USH1B and USH2A, previously published

109 Visual impairment in USH3 by Pennings et al.12 are also included for comparison. The pooled slopes indicate that the FAS deteriorated by a value of close to –1 % per year, the FFS* by a value slighty below –1.5 % per year and the combined score FVS* by a value slightly over –1.5 % per year. The parameter changes - from FFS to FFS* and from FVS to FVS* depending on the target used - did not entail substantial changes in slope for the reference groups USH1B and USH2A.

Data for the three heterozygous Finmajor-Finminor patients are shown in figure 1, but were excluded from further analysis because of the small numbers of observations. Nevertheless, it can be seen that these patients tended to show relatively poor scores for their age.

(%) FAS (%) FFS*

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80 80

60 60

40 40

20 20

0 0 10 20 30 40 50 60 10 20 30 40 50 60

(%) FVS*

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80

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20

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Figure 2. FAS, FFS* and FVS* for USH3 (solid triangles) FinMajor homozygotes. Individual longitudinal measurements connected by hairlines. Individual regression lines are included; these are bold for significant deterioration. The bold dotted line is the regression line for the cross-sectional USH3 data (Figure 1). Incidental longitudinal data of one FinMajor/FinMinor compound heterozygote are also plotted (solid squares) without a regression line.

110 Chapter 3.2

Longitudinal data Longitudinal data for USH3 are shown in Figure 2. Only one patient of the nine patients for whom longitudinal regression lines could be calculated showed significant deterioration of the FAS. Significant proportions of the patients (according to binomial statistics) showed significant deterioration of the FFS* (six of nine patients) or the FVS* (four of six). The slope of each individual longitudinal regression line was compared to the slope of the corresponding overall cross- sectional regression line (Figure 2, dotted line). Individual deterioration rates varied fairly symmetrically around the slope for the corresponding cross-sectional regression line and ranged from –2 to 0.6 for the FAS, -3.4 to 0.3 for the FFS* and between –2 to –1.2 for the FVS*.

Discussion The cross-sectional analysis in this study revealed a significant deterioration of the FAS (1.3% per year), FFS* (1.4% per year) and FVS* (1.8% per year) with advancing age for USH3 patients. This rate of deterioration in visual function was fairly similar to that of USH1B or USH2A patients. The FVS* in USH3 patients was significantly poorer than in USH2A patients; the 50% score was attained about one decade earlier in the former. Inspection of the longitudinal data showed a rate of deterioration in visual function similar to the results from the cross-sectional analysis.

The heterozygous Finmajor-Finminor patients tended to show relatively poor functional vision scores compared to the homozygous Finmajor-Finmajor patients. Because of the small number (n = 3) they were excluded from further analysis. However, this is an interesting feature because the Finmajor-Finminor patients tend to have better auditory function than homozygotes.4

Earlier studies on VA and visual field deterioration in Usher syndrome compared USH1 patients and USH2 patients.12,14-20 In general, USH2 patients showed (significantly) more favourable results than USH1 patients. Fishman et al.14 suggested that visual fields in USH1 deteriorate more rapidly than in USH2. Our data confirms that at a given age the USH2A patients score better than USH1B patients; however, a more rapid deterioration was not observed.

111 Visual impairment in USH3

The study on Dutch Usher syndrome patients found no significant difference in field scores between patients with and without cataract.12. For this reason we did not consider the issue of cataract in the analysis in our present study, although we realise that cataract could cause a difference in functional vision scores.

The previously published report on visual function loss in USH3 patients describes the course of visual handicap and typical refractive errors in USH3. Deterioration occurred below the age of 40 years, VA dropped below 0.05 (severely impaired) at the age of 37 and the visual fields were of tubular shape without any peripheral islands at the average age of 30.10 They also suggested that hypermetropia with astigmatism is pathognomonic for USH3.

As described under Patients and Methods, the standard visual field scores could not be used in the analysis of the USH3 patient data. In order to check whether we could use the V-4 target instead of the III-4 target, we used linear regression analysis (data not shown), to compare between the FFS* and FFS scores, as well as between the FVS* and the FVS data, for the Dutch Usher patients. It appeared that FFS* = FFS +14 and FVS* = FVS +14 over the whole range of scores involved. This finding implies that the changes in evaluation parameters applied did not introduce any apparent change in deterioration rate. The difference by a score of 14 should be kept in mind when comparing the present altered scores (FFS* or FVS*) to scores according to the original definitions (FFS or FVS). Thus, the FVS approximates the 50% score when FVS*=64%.

Non-linear regression analysis focussing on FVS*=64% showed that, on average, for USH3 patients the FVS attains values <50% at age >20 years, whereas this can be expected to be the case at age >31 years for USH2A patients and ages >24 years for USH1B patients. This is a clinnically important score because these patients (FVS < 50%) may no longer sufficiently benefit from vision enhancement techniques.13

It should be emphasized that most of the visual function deterioration in USH3 patients occurs after the deterioration in auditory function. Hearing deterioration is most severe during the first decades of life.4

In conclusion, the rate of deterioration of visual function in Finnish USH3 patients was fairly similar to that in Dutch USH1B or USH2A patients. At a given age, visual

112 Chapter 3.2 field impairment in USH3 patients was fairly similar to that in USH1B patients but significantly poorer than in USH2A patients.

References 1. Usher CH: On the inheritance of retinitis pigmentosa, with notes of cases. R Lond Ophthalmol Hosp Rep 1916;19:130-236. 2. Spandau UHM, Rohrschneider K. Prevalence and geographical distribution of Usher syndrome in Germany. Graefe’s Arch Clin Exp Ophthalmol 2002;240:495-498. 3. Van Camp G, Smith RJH. Hereditary Hearing loss Homepage (HHH) World Wide Web URL: http://dnalab-www.uia.ac.be/dnalab/hhh. Accessed July 2004. 4. Plantinga RF, Kleemola L, Huygen PL, Joensuu T, Sankila E-M, Pennings RJ, Cremers CW. Serial audiometry and speech recognition findings in Finnish USH3 patients. Audiol Neurootol 2005 Mar-Apr;10(2):79-89. 5. Pennings RJE, Wagenaar M, Aarem A van, Huygen PLM, Kimberling WJ, Cremers CWRJ. Hearing impairment in Usher’s syndrome. In Cremers CWRJ, Smith RJH (eds), Genetic hearing impairment. Its clinical presentations. Adv Otorhinolaryngol 2002;61:184-91. 6. Pakarinen L, Karjalainen S, Simola KOJ, Laippala P, Kaitalo H. Usher’s syndrome type 3 in Finland. Laryngoscope 1995;105:613-7. 7. Peltonen L, Jalanko A, Varilo T. Molecular genetics of the Finnish disease heritage. Hum Mol Genet 1999;8:1913-23. 8. Joensuu T, Hämäläinen R, Yuan B, Johnson C, Tegelberg S, Gasparini P, Zelante L, Pirvola U, Pakarinen L, Lehesjoki A-E, Chapelle A de la, Sankila E-M. Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3. Am J Hum Genet 2001;69:673-84. 9. Adato A, Vreugde S, Joensuu T, Avidan N, Hamalainen R, Belenkiy O, Olender T, Bonne-Tamir B, Ben-Asher E, Espinos C, Millian JM, Lehesjoki AE, Flannery JG, Avraham KB, Pietrokovski S, Sankilla EM, Beckman JS, Lancet D. USH3A transcripts encode clarin-1, a four-transmembrane- domain protein with a possible role in sensory synapses. Eur J Hum Genet 2002; 10:339-50. 10. Pakarinen L, Tuppurainen K, Laippala P, Mantyjarvi M, Puhakka H. The ophthalmological course of Usher syndrome type III. Int Ophthalmol 1995-96;19(5):307-11. 11. Auffarth GU, Tetz MR, Krastel H, Blankenagel A, Volcker HE. Cataracta complicata bei ver- schiedenen Formen der Retinitis Pigmentosa. Art und Häufigkeit. Ophthalmologe 1997;94:642- 646. 12. Pennings RJ, Huygen PL, Orten DJ, Wagenaar M, van Aarem A, Kremer H, Kimberling WJ, Cremers CW, Deutman AF. Evaluation of visual impairment in Usher syndrome 1b and Usher syndrome 2a. Acta Ophthalmol Scand 2004;82(2):131-9. 13. American Medical Association. Chapter 12 The visual system. In: Cocchiarella L & Anderson GBJ (eds.) Guides to the Evaluation of Permanent Impairment, 5th edition. Chicago. American Medical Association Press: 277-304, 2001. 14. Fishman GA, Kumar A, Joseph ME, Torok N, Anderson RJ. Usher’s syndrome. Ophthalmic and neuro-otologic findings suggesting genetic heterogeneity. Arch Ophthalmol 1983;101:1367-1374. 15. Piazza L, Fishman GA, Farber M, Derlacki D, Anderson RJ. Visual acuity loss in patients with Usher’s syndrome. Arch Ophthalmol 1986;104:1336-1339.

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16. van Aarem A, Wagenaar M, Pinckers AJLG, Huygen PLM, Bleeker-Wagemakers EM, Kimberling WJ, Cremers CWRJ. Ophthalmological findings in Usher syndrome type 2A. Ophthalmic Genet 1995;16:151:158. 17. Fishman GA, Anderson RJ, Lam BL, Derlacki DJ. Prevalence of foveal lesions in type 1 and type 2 Usher’s syndrome. Arch Ophthalmol 1995;113:770-773. 18. Edwards A, Fishman GA, Anderson RJ, Grover S, Derlacki DJ. Visual acuity and visual field impairment in Usher syndrome. Arch Ophthalmol 1998;116:165-168. 19. Seeliger M, Pfister M, Gendo K, Paasch S, Apfelstedt-Sylla E, Plinkert P, Zenner H-P, Zrenner E. Comparative study of visual, auditory and olfactory function in Usher syndrome. Graefe’s Arch Clin Exp Ophthalmol 1999;237:301-307. 20. Tsilou ET, Rubin BI, Caruso RC, Reed GF, Pikus A, Hejtmancik JF, Iwata F, Redman JB, Kaiser- Kupfer MI. Usher syndrome clinical types I and II: Could ocular symptoms and signs differentiate between the two types? Acta Ophthalmol Scand 2002;80:196-201.

114 Chapter 4

Hearing impairment in genotyped Wolfram syndrome patients

Rutger F. Plantinga, Ronald J.E. Pennings, Patrick L.M. Huygen, Rocco Bruno, Phillipp Eller, Thimothy G. Barrett, Bernard Vialettes, Veronique Paquis-Fluklinger, Fortunato Lombardo, Cor W.R.J. Cremers. Submitted. Abstract Wolfram syndrome is a progressive neurodegenerative syndrome characterized by the features Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness (DIDMOAD). This multicentre study was conducted to analyse the audiometric data of 23 genotyped Wolfram syndrome patients with sensorineural hearing impairment. In all patients at least one mutation was identified in WFS1. Pure tone threshold data were used for cross-sectional analysis in subgroups of patients: male/female, young (<16 years)/old (19-25 years) and previously reported Dutch data/present additional multi-centre data. With one exception, all subgroups showed a fairly similar type of hearing impairment with, on average, thresholds of about 25 dB at 0.25-1 kHz, gently sloping downwards to about 60 dB at 8 kHz. The subgroup of previously reported Dutch female patients showed an exceptionally high degree of hearing impairment. We could find no other reason than gender for this exception, which was probably not related to the types of WFS1 mutations identified in (most of) these females. Chapter 4.1

Introduction Wolfram syndrome is a progressive neurodegenerative syndrome characterized by the features Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness (DIDMOAD). It is an autosomal recessive inherited syndrome that was first described by Wolfram and Wagner in 1938.1 Wolfram syndrome is rare with an estimated prevalence of 1:770,000.2 Minimal clinical diagnostic criteria are juvenile-onset diabetes mellitus and optic atrophy both mainly manifesting in the first decade of life. In addition to the classical four features, patients can develop renal tract abnormalities, apnoea, cerebellar ataxia, behavioral and psychiatric illness, gastro- intestinal dysmotility and primary gonadal atrophy. In general, Wolfram syndrome is a progressive neurodegenerative disorder that affects central, peripheral and neuroendocrine components of the nervous system leading to death before or by mid-life.3

Genetic studies have identified WFS1, located on chromosome 4p16.3, as the gene involved in Wolfram syndrome.4,5 WFS1 codes for wolframin, a transmembrane protein comprising 890 amino acids.5 Wolframin is expressed in the endoplasmatic reticulum of a number of cell types in heart, lung, brain and pancreas.5,6 Immunostaining in developing murine inner ear showed that wolframin localizes to the canalicular reticulum, a specialized form of endoplasmatic reticulum, and is involved in endoplasmatic reticulum stress responses, including apoptosis.7-9 The canalicular reticulum has been identified in various types of inner ear cells, including vestibular and cochlear hair cells, spiral ganglion cells, Deiter’s cells and cells in the stria vascularis and is suggested to be involved in intercellular ion transport, an important process in the physiology of the inner ear.3,7 However, the exact role and function of wolframin remain unclear.

Mutations in WFS1 are not only responsible for the autosomal recessive Wolfram syndrome, but also for an autosomal dominant form of non-syndromic sensorineural hearing impairment designated DFNA6/14/38.10,11 Wolfram syndrome is characterized by high-frequency sensorineural hearing impairment, whereas DFNA6/14 is characterized by low-frequency sensorineural hearing impairment.12,13 Hearing impairment in DFNA6/14 can be either progressive or non-progressive.13 Mutation analysis of WFS1 in families with DFNA6/14 revealed that the identified pathogenic mutations in this disorder all are small non-inactivating (missense) mutations, whereas most of the pathogenic mutations in Wolfram syndrome

117 Wolfram syndrome patients are inactivating mutations.14 Of note is that Eiberg et al. described a family with a missense mutation in WFS1 showing dominant optic atrophy associated with hearing impairment and impaired glucose regulation.15

In 2004, Pennings et al. reported on 11 Dutch Wolfram syndrome patients with confirmed mutations in WFS1. They concluded that progression in hearing impairment mainly occurred in the mid- and high-frequency range. In addition, they observed that the hearing impairment in 5 female patients with Wolfram syndrome was substantially worse when compared to the hearing impairment in 4 male patients.16 They hypothesised that this sex-related difference in hearing impairment might be explained by the involvement of estrogen.16 Two remaining older patients had a mild phenotype caused by two missense mutations in WFS1. Realizing that the sex-related hearing impairment finding is only based on data collected in one centre and on a small number of patients, we felt that it merits re-evalution in a multi- centre study. Apart from that, collecting additional data allows us to further outline the feature of sensorineural hearing impairment (SNHI) in Wolfram syndrome in terms of presence and degree of progression and degree of variability. Therefore, this multi-centre study was performed in close collaboration with co-authors of previous clinical and genetic reports on Wolfram syndrome in order to obtain audiometric data from genotyped Wolfram syndrome patients.

Patients and methods This multi-centre study included 23 patients with Wolfram syndrome from whom audiograms had been obtained and were judged to show clinically relevant SNHI. All of them were previously diagnosed with Wolfram syndrome and in each of them at least one mutation was identified in WFS1. After written informed consent, audiological data were retrieved and, when necessary, patients underwent additional audiometric evaluation in the respective local centres. Table 1 provides an overview of the relevant data pertaining to the included subjects. In all patients, only inactivating mutations in WFS1 were identified. For the sake of the Discussion, we also performed an extensive study of literature, covering reports that appeared before and after the review by Cremers et al. to trace audiograms of Wolfram syndrome patients.12

118 Chapter 4.1

Table 1. Details of included Wolfram syndrome patients.

Patient Origin Family Gender Mutation(s) Ref. no. Dutch WF1 1, 2 F, M Splice site (460+1G>A; 460+1G>A) 17 WF2 3 F Y528fsX542 17 WF3 4, 5 M, F Y508-L512del; Y508-L512del 17 WF4 6 F Y508-L512del; V412fsX440 17 WF6 7 M V509fsX517; V509fsX517 17 WF10 8, 9 M, F Q667X; V142fsX251 17 Austria 1 10 M L347fsX542; L347fsX542 18 United Kingdom - 11 M Q112X; Q112X - France WS2 12, 13 M, F p.W540del; p.W540del 19 WS4 14 F p.W371X; p.W371X 19 WS7 15 M p.Y291X; p.S411fsX541 19 WS9 16 F p.V142fsX251; p.L554_G562del 19 Italy WS 17 – 23 3M, 4F Y454X; Y454X 20

Audiometry comprised measurement in a sound-treated room of pure tone thresholds at octave frequencies 0.25 – 8 kHz for air conduction and bone conduction according to common clinical standards. Bone conduction levels were evaluated only to exclude .

Statistical analyses Pure tone threshold data (binaural mean air conduction threshold) were used for cross-sectional analysis in subgroups of patients established by distinguishing 3 types of dichotomies: male/female, young/old and Dutch collective data16/present additional multi-centre data. After a first inspection of the collective data, we decided to classify patients aged 16 years or younger as “young” patients and those aged 19-25 years as “old” patients. Three of the female patients of the study by Pennings et al. had also been evaluated at higher ages (27-35 years); of the additionally collected cases, only two had been measured at ages older than 25 years (32 and 37 years). The cases only measured at age > 25 years were excluded from the present study, however, one case (no. 7) showed a close-to-normal threshold at 0.25- 4 kHz at age 28.16 years and was therefore included in the age group of 19-25 years.

119 Wolfram syndrome

A <16 yrs 19-25 yrs

dB HL Pat 4 dB HL Pat 2 dB HL Pat 7 dB HL Pat 2 -10 -10 -10 -10 0 14,51 0 15,70 0 28,16 0 20 20 20 20 22,06 40 40 40 40

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.25.51 24 8kHz .25.51 24 8 kHz .25.51 248 kHz .25 .5 1 24 8 kHz Male dB HL Pat 8 dB HL Pat 8 -10 -10 0 11,84 0 20 20

40 40 20,40 22,61 60 60 24,69 80 80

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dB HL Pat 6 dB HL Pat 5 dB HL Pat 6 dB HL Pat 5 -10 -10 -10 -10 0 8,91 0 10,33 0 0 20 12,45 20 13,48 20 20 40 40 40 40 19,23 21,97 60 60 60 60 25,08 80 80 80 80

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120 120 120 120 Female .25.51 24 8kHz .25.51 24 8kHz .25.51 24 8kHz .25 .5 1 24 8kHz

dB HL Pat 1 dB HL Pat 9 dB HL Pat 3 dB HL Pat 9 -10 -10 -10 -10 0 12,27 0 7,98 0 20,65 0 20 15,78 20 11,27 20 20 40 40 40 40 20,58 60 60 60 60 22,54 80 80 80 80

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.25.51 24 8kHz .25.51 24 8kHz .25.51 24 8kHz .25 .5 1 24 8kHz

120 Chapter 4.1

B < 16 yrs 19-25 years

dB HL Pat 11 dB HL Pat 20 dB HL Pat 10 dB HL Pat 12 -10 -10 -10 -10 0 8,10 0 9,20 0 23.69 0 22,60 20 12,77 20 20 20 40 14,40 40 40 40

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Figure 1 (A, B). Individual binaural mean air conduction thresholds in 8 subgroups of genotyped patients: (A), Dutch patients; (B), additional patients. (A) and (B) both include 4 panels. Panels on the left cover ages of < 16 years, panels on the right cover ages 19-25 years. Top panels include male patients, bottom panels female patients. Of the Dutch patients only the audiograms obtained within the intended age range are shown in the individual, small panels. Age (years) is included in the symbol key to each small panel.

121 Wolfram syndrome

Several of the Dutch patients had been measured at various ages. Their audiogram data were included where possible in order to establish the “best possible” estimate of the typical audiogram in relation to age. We realised, however, that different samples bearing on the same individuals are not stochastically independent and should therefore not be formally compared in cross-sectional analyses. Statistical tests were only performed on stochastically independent samples (Results). One- way analysis of variance was performed to compare across-individuals mean pure tone thresholds at each frequency across a number (n>2) of subgroups. Student’s t test was used to compare such mean values between relevant pairs of subgroups; this test included Welch’s correction if Bartlett’s test detected unequal variances. The level of significance used was p = 0.05 in each test.

Results Figure 1 shows the individual audiometric data for air conduction obtained for all genotyped patients with Wolfram syndrome, arranged in 8 subgroups. None of the examined individuals showed an air-bone gap. For each of the 8 panels shown in Figure 1 (A, B) a composite audiogram was drawn, which only included the last-visit audiogram within the predetermined age range, to check that pooling was allowed. This was indeed the case (data not shown) and the “mean audiogram” resulting from averaging the included audiograms is shown in each of the corresponding panels of Figure 2 (A, B). Statistical tests (Methods) demonstrated no significant differences in age between any of the subgroups that were intended to cover the same age range. There was no significant difference in mean threshold found at any frequency across any of the 4 subgroups pertaining to the additional cases (Figure 2B). We therefore conclude that the additional patients do not disclose a substantial difference in hearing impairment between the sexes or substantial progression in hearing impairment with advancing age. No significant difference in mean threshold was observed at any frequency within the subgroups of male patients pertaining to any of the different subgroups (Dutch/additional patients, age <16 years/age 19-25 years). This finding demonstrates that the males of the Dutch subgroup and the additional present males showed a fairly similar degree of hearing impairment.

A significant difference in mean threshold was found at ages <16 years (left panels of Figure 2A and Figure 2B) between the Dutch females (Fig. 2A, bottom panels) and the additional females (Figure 2B, bottom panels) at the frequencies 1-8 kHz (p=

122 Chapter 4.1

0.005-0.03 in Student’s t-test). At ages 19-25 years (right panels of Figure 2A and Figure 2B), there was only a significant difference in mean threshold found at 0.5 kHz (p=0.02) when testing between the subgroups relating to the Dutch and the additional females, respectively. At the adjacent frequencies 0.25, 1 and 2 kHz, however, Student’s t test produced p values of ~0.08 each; clearly, such a constellation of findings cannot be attributed to chance alone (binomial statistics). We conclude that the Dutch females showed a substantially higher degree of SNHI than not only the Dutch males, as was concluded in the previous study, but also a higher degree of SNHI than the additional collected females in the present study.16 It should be noted that, although these females did show significant progression according to the individual longitudinal analyses of our previous report,16 this finding did not appear from the previous16 or the present cross-sectional analysis (Figure 2A, bottom pannel). This apparent discrepancy may be due to the relative large intersubject variability shown by the cross-sectional threshold data involved (Figure 2A). A B

d B HL M: <16 yr d B HL M: 19-25 yr dB HL M: <16 yr dB HL M: 19-25 yr -10 -10 -10 -10 0 0 0 0 20 20 20 20

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Figure 2. Mean audiograms for the same subgroups as shown in Fig. 1: (A), Dutch patients; (B), additional patients. Each separate, small panel includes a legend indicating sex (M, male; F, female) and age range (0-16 years; 19-25 years). Bars above and below symbol are 1 SD each.

123 Wolfram syndrome

Given the above described observations, it is clear that the subgroup of Dutch females distinguished itself from the other subgroups of patients by its substantially higher degree of SNHI. All additional patients (age <24 years) can be simultaneously characterized in a satisfactory way by one “mean” audiogram with a threshold of about 25 dB at 0.25-1 kHz, gently sloping down to about 60 dB at 8 kHz. Implicit to this statement is the notion that none of these subgroups showed substantial progression in SNHI with advancing age, at least throughout the first 3 decades of life.

We focused on using mutation data (Table 1) in pairwise comparisons by threshold (Figure 1). Controlling for mutation can be performed by comparisons between sibs having the same mutations. The Italian sibs, patients 17-23, showed a remarkable similarity of hearing thresholds regardless of gender and age (Figure 1B). The French sibs (Figure 1B), patients 12 (male) and 13 (female), also showed similar thresholds, however, the female sib was still young (12 years). Inter-sib comparisons within the group of Dutch patients are especially intriguing because of the remarkbly poor thresholds in the female patients. The pair of sibs, patients 1 (female) and 2 (male), reflect the previously described gender difference.16 Patient 1 showed a relatively poor threshold even among the young Dutch female patients (Fig. 1A). Remarkably, also her sib, patient 2, seemed to show a relatively poor threshold among the Dutch males. The sibs patients 4 (male) and 5 (female) showed fairly similar thresholds at age < 16 years. At a more advanced age, patient 5 (female) showed considerable threshold deterioration, however, we had no threshold data for her sib, patient 4 (male), at a similar age. The pair of sibs patients 8 (male) and 9 (female), allowed for comparisons within both age categories: there was no substantial difference between them at age < 16 years, whereas a clear difference was shown at age 19-25 years (Fig. 1A), associated with the substantial deterioration shown by patient 9 (female), an effect that might be attributed to a gender effect.

Discussion This study comprises a detailed audiometric analysis of genotyped Wolfram syndrome patients. As described above, the hearing impairment in Wolfram syndrome patients showed a fairly similar spectrum of SNHI in different subgroups composed by dichotomising the total patient group by gender, age class and origin (Dutch patients included in previous study vs additional patients in present study). However, the subgroup of previously examined Dutch females stands out. Their

124 Chapter 4.1 hearing impairment is much more severe when compared to the other subgroups. This finding seems difficult to explain. However, keeping in mind that any difference in threshold may be due to differences in mutation and age, and perhaps also gender, we focused on comparing thresholds while controlling for mutations (Figure1). The factors mutation(s) and age could be simultaneously controlled for only in a minority of cases. Pairwise threshold comparisons between the Dutch sibs, patients 8 and 9, reflected the previously stipulated gender difference that became prominent at a more advanced age. Multiple comparisons were possible within the Italian sibship, patients 17-23 (Figure 1B), where no such difference could be demonstrated. However, it should be emphasized that the maximum ages covered by the Italian sibship were still < 22 years. Therefore, the possibility cannot be excluded that the gender effect previously described for the Dutch patients, can be substantiated in future studies, provided that sufficient data can be collected from fully genotyped patients covering more advanced ages and allowing for adequate controlling for mutations in the relevant analyses. In an effort to extend on the current data, a literature search was performed in order to obtain previously reported audiometric data. Figure 3 shows the audiogram data of Wolfram syndrome patients with SNHI that could be retrieved.

Molecular genetic analysis was not reported for any of these cases. Their ages were in the range of 10-14 years. There was no apparent difference between the male (top panel in Figure 3) and the female (bottom panel) patients. For the sake of comparison, we plotted the mean audiogram obtained for the present additional, genotyped patients (see Figures 1B and 2B) aged <16 years (dotted line). All literature cases except one (Pat 27 in Figure 3) had their threshold within 20 dB of this mean threshold. It seems that our current audiometric data, with the exclusion of the Dutch female subgroup, is representative for hearing impairment in Wolfram syndrome patients.

Accepting the latter exclusion, we conclude that this unique collection of audiometric data from the additional genotyped Wolfram syndrome patients shows no significant sex-related differences, no substantial progression in SNHI with advancing age and that their hearing impairment can be described by an audiogram with a threshold of about 25 dB at 0.25-1 kHz, gently sloping downwards to about 60 dB at 8 kHz. It should be emphasized that the lack of gender-related differences in threshold may be explaned by a general lack of cases of sufficiently advanced age.

125 Wolfram syndrome

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Figure 3. Individual audiograms for the Wolfram syndrome patients without genotype data whose audiogram could be retrieved from literature (patients 2421, 25 and 2622, 2723, 28 and 2924, 30 and 3125, and 3226). Age 10-14 years. Top panel, male patients; bottom panel, female patients. Same symbols and legends as in Figure 1; the dotted line indicates the mean audiogram for the present additional, genotyped patients aged < 16 years.

126 Chapter 4.1

References 1. Wolfram DJ, Wagner HP. Diabetes mellitus and simple optic atrophy among siblings: report of four cases. Proc Mayo Clin 1938;13:242-250. 2. Barrett TG, Bundey SE, Macleod AF. Neurodegeneration and diabetes: UK nationwide study of Wolfram (DIDMOAD) syndrome. Lancet 1995;346:1458-1463. 3. McHugh RK, Friedman RA. Genetics of hearing loss: Allelism and modifier genes produce a phenotypic continuum. Anat Rec A Discov Mol Cell Evol Biol 2006;288:370-381. 4. Inoue H, Tanizawa Y, Wasson J, Behn P, Kalidas K, Bernal-Mizrachi E, Mueckler M, Marshall H, Donis-Keller H, Crock P, Rogers D, Mikuni M, Kumashiro H, Higashi K, Sobue G, Oka Y, Permutt MA. A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome). Nat Genet 1998;20:143-148. 5. Strom TM, Hortnagel K, Hofmann S, Gekeler F, Scharfe C, Rabl W, Gerbitz KD, Meitinger T. Diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD) caused by mutations in a novel gene (wolframin) coding for a predicted transmembrane protein. Hum Mol Genet 1998;7:2021-2028. 6. Takeda K, Inoue H, Tanizawa Y, Matsuzaki Y, Oba J, Watanabe Y, Shinoda K, Oka Y. WFS1 (Wolfram syndrome 1) gene product: predominant subcellular localization to endoplasmic reticulum in cultured cells and neuronal expression in rat brain. Hum Mol Genet 2001;10:477- 484. 7. Cryns K, Thys S, Van Laer L, Oka Y, Pfister M, Van Nassauw L, Smith RJH, Timmermans JP, Van Camp G. The WFS1 gene, responsible for low frequency sensorineural hearing loss and Wolfram syndrome, is expressed in a variety of inner ear cells. Histochem Cell Biol 2003;119:247-256. 8. Yamada T, Ishihara H, Tamura A, Takahashi R, Yamaguchi S, Takei D, Tokita A, Satake C, Tashiro F, Katagiri H, Aburatani H, Miyazaki J, Oka Y. WFS1-deficiency increases endoplasmic reticulum stress, impairs cell cycle progression and triggers the apoptotic pathway specifically in pancreatic beta-cells. Hum Mol Genet 2006;15:1600-1609. 9. Riggs AC, Bernal-Mizrachi E, Ohsugi M, Wasson J, Fatrai S, Welling C, Murray J, Schmidt RE, Herrera PL, Permutt MA. Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis. Diabetologia 2005;48:2313-2321. 10. Bespalova IN, Van Camp G, Bom SJH, Brown DJ, Cryns K, DeWan AT, Erson AE, Flothmann K, Kunst HPM, Kurnool P, Sivakumaran TA, Cremers CWRJ, Leal SM, Burmeister M, Lesperance MM. Mutations in the Wolfram syndrome 1 gene (WFS1) are a common cause of low frequency sensorineural hearing loss. Hum Mol Genet 2001;10:2501-2508. 11. Young TL, Ives E, Lynch E, Person R, Snook S, MacLaren L, Cater T, Griffin A, Fernandez B, Lee MK, King MC. Non-syndromic progressive hearing loss DFNA38 is caused by heterozygous missense mutation in the Wolfram syndrome gene WFS1. Hum Mol Genet 2001;10:2509-2514. 12. Cremers CWRJ, Wijdeveld PGAB, Pinckers AJLG. Juvenile diabetes mellitus, optic atrophy, hearing loss, diabetes insipidus, atonia of the urinary tract and bladder, and other abnormalities (Wolfram syndrome). A review of 88 cases from the literature with personal observations on 3 new patients. Acta Paediatr Scand Suppl 1977;1-16.

127 Wolfram syndrome

13. Pennings RJE, Bom SJH, Cryns K, Flothmann K, Huygen PLM, Kremer H, Van Camp G, Cremers CWRJ. Progression of low-frequency sensorineural hearing loss (DFNA6/14-WFS1). Arch Otolaryngol Head Neck Surg 2003;129:421-426. 14. Cryns K, Pfister M, Pennings RJE, Bom SJH, Flothmann K, Caethoven G, Kremer H, Schatteman I, Koln KA, Toth T, Kupka S, Blin N, Nurnberg P, Thiele H, Van de Heyning PH, Reardon W, Stephens D, Cremers CWRJ, Smith RJH, Van Camp G. Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations. Hum Genet 2002;110:389-394. 15. Eiberg H, Hansen L, Kjer B, Hansen T, Pedersen O, Bille M, Rosenberg T, Tranebjaerg L. Autosomal dominant optic atrophy associated with hearing impairment and impaired glucose regulation caused by a missense mutation in the WFS1 gene. J Med Genet 2006;43:435-440. 16. Pennings RJE, Huygen PLM, van den Ouweland JM, Cryns K, Dikkeschei LD, Van Camp G, Cremers CWRJ. Sex-related hearing impairment in Wolfram syndrome patients identified by inactivating WFS1 mutations. Audiol Neurootol 2004;9:51-62. 17. van den Ouweland JM, Cryns K, Pennings RJE, Walraven I, Janssen GM, Maassen JA, Veldhuijzen BF, Arntzenius AB, Lindhout D, Cremers CWRJ, Van Camp G, Dikkeschei LD. Molecular characterization of WFS1 in patients with Wolfram syndrome. J Mol Diagn 2003;5:88-95. 18. Eller P, Foger B, Gander R, Sauper T, Lechleitner M, Finkenstedt G, Patsch JR. Wolfram syndrome: a clinical and molecular genetic analysis. J Med Genet 2001;38:E37. 19. Giuliano F, Bannwarth S, Monnot S, Cano A, Chabrol B, Vialettes B, Delobel B, Paquis-Flucklinger V. Wolfram syndrome in French population: characterization of novel mutations and polymorphisms in the WFS1 gene. Hum Mutat 2005;25:99-100. 20. Lombardo F, Chiurazzi P, Hortnagel K, Arrigo T, Valenzise M, Meitinger T, Messina MF, Salzano G, Barberi I, De Luca F. Clinical picture, evolution and peculiar molecular findings in a very large pedigree with Wolfram syndrome. J Pediatr Endocrinol Metab 2005;18:1391-1397. 21. Barjon P, Lestradet H, Labauge R. Atrophie optique primitive et surdité neurogène dans le diabète juvénile (A propos de trois observations). Presse Med 1964;72:983-986. 22. Laffay G, Lestradet H. Diabète juvénile et atrophie optique primitive. Sem Hop 1974;50:127-141. 23. Megighian D, Savastano M. Wolfram syndrome. Int J Pediatr Otorhinolaryngol 2004;68:243- 247. 24. Higashi K. Otologic findings of DIDMOAD syndrome. Am J Otol 1991;12:57-60. 25. Sauer H, Chuden H, Gottesburen H, Schmitz-Valckenberg P, Seitz D. Familiäres Vorkommen von Diabetes mellitus, primärer Opticusatrophie und Innenohrschwerhörigkeit. Dtsch Med Wochenschr 1973;98:243-255. 26. Lin CH, Lee YJ, Huang CY, Shieh JW, Lin HC, Wang AM, Shih BF. Wolfram (DIDMOAD) syndrome: report of two patients. J Pediatr Endocrinol Metab 2004;17:1461-1464.

128 Chapter 5

General discussion

Chapter 5

This thesis on hereditary hearing impairment elucidates some clinical and genetic aspects of DFNA8/12 (Chapter 2), Usher syndrome type III (Chapter 3) and Wolfram syndrome (Chapter 4).

Chapter 2 discusses DFNA8/12(TECTA) and provides an overview of the hereditary and audiological aspects of this type of hearing impairment. A general review of DFNA8/12 is provided in Chapter 2.1 (in Dutch). In Chapter 2.2 we report on a novel TECTA mutation, p.R1890C, found in a Dutch family with nonsyndromic autosomal dominant sensorineural hearing impairment. In early life a, presumably congenital, hearing impairment occurred in the midfrequency range, amounting to about 40 dB at 1 kHz. The missense mutation is located in the zona pellucida (ZP) domain of D- tectorin. Mutations affecting the ZP domain of Į-tectorin are significantly associated with midfrequency hearing impairment.

Since 2003, one of the foci of our otogenetic projects has been on families with a midfrequency type of sensorineural hearing impairment. The DFNA8/12(TECTA) family described above is the first large midfrequency hearing impaired family that we studied, with linkage to the DFNA8/12 locus. Thus far, we could not find linkage in three additional large midfrequency hearing impaired families. It will be interesting to see whether in these other midfrequency families the mutations will prove to be on the same locus, the locus of another type of DFNA, or perhaps a new locus.

In Chapter 2.3 we present the results of an extensive audiological evaluation of five selected members from the DFNA8/12(TECTA) family. The results were similar to the specific audiological features previously obtained from a Dutch DFNA13(COL11A2) family with midfrequency sensorineural hearing impairment.1 In this DFNA13(COL11A2) family the mutations lead to an abnormal type XI collagen Į2- chain, resulting in collagen fibril (type II collagen) disorganization in the tectorial membrane.2 It appears that these types of midfrequency sensorineural hearing impairment, caused by a defect in the tectorial membrane, may be characterized as a cochlear type of conductive hearing impairment. This type of audiological testing could lead to a new field of topo-audiological research, facilitated by otogenetic family studies. Eventually, after further research, this type of testing might be useful in determining the location of the defect.

131 General discussion

In children and young adults affected with midfrequency hearing impairment, it can easily go unnoticed, because of the good speech reception scores associated with the relatively favourable thresholds in the high frequencies. This situation is comparable to speech perception in noise for normal hearing subjects, where the emphasis lies on the perception of consonants in the 2-4 kHz range. Providing adequate amplification can easily compensate the attenuation of sound in the inner ear. Hearing aid fitting may restore hearing close to normal. However, the favourable thresholds for the low frequencies provide a challenge for optimal hearing aid fitting, as the occlusion effect introduced by an earmold can put too much emphasis on the low frequencies. Fitting with an open earmold may be the best solution. In some cases the presence of progression can be troublesome. We consider it worthwhile to continue our focus on midfrequency sensorineural hearing impairment to support affected subjects needing specific audiological support and guidance.

In Chapter 3 the phenotype of Usher syndrome type III (USH3) is discussed in great detail. In the otogenetic literature a comprehensive report on this phenotype was still lacking. Outside Finland USH3 is quite rare.3,4 The high prevalence of USH3 in Finland is caused by the Finnish Disease Heritage, a collection of nearly 40 rare hereditary diseases over-represented in Finland.5,6 In order to study this phenotype we closely collaborated with Finnish colleagues, who were the first to identify this type of Usher syndrome.

Chapter 3.1 describes the audiometric features in genotyped Finnish USH3 patients. The group of Finnish USH3 patients demonstrated an impressively wide spectrum of progressive sensorineural hearing impairment: from normal to moderate USH2A- like hearing impairment at a young age to profound or even USH1B-like hearing impairment at a more advanced age. The highest progression was seen during the first two decades of life, gradually slowing down with further ageing. This type of nonlinear progression may be unique amongst the Usher syndromes. Analysis of speech recognition scores in relation to age also disclosed a highly variable deterioration of speech recognition. The USH2A patients showed much more uniformity in speech recognition scores related to age and the level of SNHI than the present USH3 patients. However, the performance-impairment plots, including the estimated onset level and deterioration gradient, were fairly similar in USH3 and USH2A patients.

132 Chapter 5

Later, Sadeghi et al. published a phenotypical report on USH3 with quite similar results and in addition they observed that vestibular function was variable, ranging from normal to vestibular dysfunction.7 Because of the high degree of variability in threshold features and vestibular function, it seems preferable to base the diagnosis of USH3 on the genotype obtained through mutation analysis. Outside the Scandinavian countries USH3 is rare and patients with USH3 may in fact be regarded as USH1 or USH2 patients due to similarity with these clinical types or unfamiliarity with the USH3 phenotype. The recent development and availability of the Usher syndrome mutation microarray assists us to identify USH3 patients outside the Scandinavian countries in a cost-effective way. Routine screening of mutations in all currently known Usher syndrome genes are offered through this micro-array and this is very helpful in the genetic counselling of not only USH3 patients but of all patients with Usher syndrome.8

Chapter 3.2 evaluates visual impairment in Finnish genotyped Usher syndrome type III patients and provides a comparison with visual impairment in patients with other types of Usher syndrome (USH1B and USH2A).9 Most of the visual function deterioration in USH3 patients occurred after the deterioration in auditory function. The rate of deterioration in visual function in Finnish USH3 patients was fairly similar to that in Dutch USH1B or USH2A patients. However, analysis focussing on the clinically important functional vision score < 50% showed that this score, on average, in USH3 patients is reached at age >20 years, whereas this can be expected to be the case at age >31 in USH2A patients and age >24 in USH1B patients. Only homozygous Finmajor-Finmajor USH3 patients were included in our analysis. Surprisingly, the available data of compound heterozygous Finmajor-Finminor patients tended to show relatively poor functional vision scores compared to the homozygote patients. This is an interesting feature because the Finmajor-Finminor patients tend to have a better auditory function than homozygote patients.

Given the previous observations, it can be quite challenging to clinically classify the various types of Usher syndrome. The only two features that stand out are the degree of hearing impairment and vestibular function. However, in order to differentiate between the three types it can be necessary to have sufficient follow- up data available. The results of the present genotype-phenotype studies contribute to the worldwide counselling of Usher syndrome patients and their relatives and can be of great importance for future research. Gene therapy is one of the developments

133 General discussion in Usher syndrome research that may lead to a possible curative therapeutic intervention for this serious condition that affects both vision and hearing. Several research groups are currently developing gene therapy for genetic subtypes of Usher syndrome. A possible advantage for USH3 patients is the fact that the USH3 gene is relatively small and can thus be placed much more easily in a gene therapy vector like the adeno-associated virus than the other larger Usher syndrome genes.10 Future therapeutic studies of gene therapy in USH3 patients will focus on the effect of the treatment. This will be done by studying the phenotype after intervention and correlate this to the general deterioration known for a specific subset of patients. In addition, this is one of the main reasons why studies of the phenotype are so important for these patients.

Chapter 4 focuses on the phenotypical aspect of hearing impairment in genotyped Wolfram syndrome patients. An international multi-centre study was conducted to evaluate the degree and progression of sensorineural hearing impairment in Wolfram syndrome patients. Furthermore, we examined whether or not the previously reported gender-related difference existed.11 With the exception of the group of previously reported Dutch female patients, all subgroups showed a fairly similar type of hearing impairment with, on average, thresholds of about 25 dB at 0.25-1 kHz, gently sloping downwards to about 60 dB at 8 kHz.11 This degree of hearing impairment was quite similar to the audiometric data of clinically diagnosed Wolfram syndrome patients obtained from literature.12-17 It seemed that our audiometric data, with the exclusion of the Dutch female subgroup, is representative for hearing impairment in Wolfram syndrome patients.11 Accepting the latter exclusion, our collected audiometric data from genotyped Wolfram syndrome patients showed no substantial progression in sensorineural hearing impairment with advancing age and no significant gender-related differences. However, the possibility of gender-related differences in threshold at more advanced ages could not be ruled out due to a general lack in cases of sufficiently advanced age. The key to further clarifying the phenotypical aspect of hearing impairment in Wolfram syndrome, as well as other features in these kinds of rare syndromes lies, without doubt, in the continuing efforts to cooperate on an international level. To examine further the hearing impairment in Wolfram syndrome patients, it will be worthwhile to perform prospectively annual pure tone audiometry in the currently known Wolfram syndrome patients. Analyses of these

134 Chapter 5 data may explain why there is a difference between the Dutch female patients with Wolfram syndrome and the remaining patients.

References 1. De Leenheer EMR, Bosman AJ, Kunst HPM, Huygen PLM, Cremers CWRJ. Audiological characteristics of some affected members of a Dutch DFNA13/COL11A2 family. Ann Otol Rhinol Laryngol 2004;113:922-929. 2. McGuirt WT, Prasad SD, Griffith AJ, Kunst HPM, Green GE, Shpargel KB, Runge C, Huybrechts C, Mueller RF, Lynch E, King MC, Brunner HG, Cremers CWRJ, Takanosu M, Li SW, Arita M, Mayne R, Prockop DJ, Van Camp G, Smith RJH. Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13). Nat Genet 1999;23:413-419. 3. Aller E, Jaijo T, Oltra S, Alio J, Galan F, Najera C, Beneyto M, Millan JM. Mutation screening of USH3 gene (clarin-1) in Spanish patients with Usher syndrome: low prevalence and phenotypic variability. Clin Genet 2004;66:525-529. 4. Pakarinen L, Karjalainen S, Simola KO, Laippala P, Kaitalo H. Usher's syndrome type 3 in Finland. Laryngoscope 1995;105:613-617. 5. Norio R, Nevanlinna HR, Perheentupa J. Hereditary diseases in Finland; rare flora in rare soul. Ann Clin Res 1973;5:109-141. 6. Norio R. The Finnish Disease Heritage III: the individual diseases. Hum Genet 2003;112:470-526. 7. Sadeghi M, Cohn ES, Kimberling WJ, Tranebjaerg L, Moller C. Audiological and vestibular features in affected subjects with USH3: a genotype/phenotype correlation. Int J Audiol 2005;44:307-316. 8. Cremers FP, Kimberling WJ, Kulm M, de Brouwer A, van Wijk E, Te Brinke H, Cremers CWRJ, Hoefsloot LH, Banfi S, Simonelli F, Fleischhauer JC, Berger W, Kelley PM, Haralambous E, Bitner- Glindzicz M, Webster AR, Saihan Z, De Baere E, Leroy BP, Silvestri G, McKay G, Koenekoop RK, Millan JM, Rosenberg T, Joensuu T, Sankila EM, Weil D, Weston MD, Wissinger B, Kremer H. Development of a genotyping Microarray for usher syndrome. J Med Genet 2006; Nov 14 [Epub ahead of print]. 9. Pennings RJE, Huygen PLM, Orten DJ, Wagenaar M, Van Aarem A, Kremer H, Kimberling WJ, Cremers CWRJ, Deutman AF. Evaluation of visual impairment in Usher syndrome 1b and Usher syndrome 2a. Acta Ophthalmol Scand 2004;82:131-139. 10. Flannery, J. G. Development of an animal model of Usher syndrome type III. First International Symposium on Usher syndrome and related Disorders. Omaha, Nebraska. 2006. Abstract. 11. Pennings RJE, Huygen PLM, van den Ouweland JM, Cryns K, Dikkeschei LD, Van Camp G, Cremers CWRJ. Sex-related hearing impairment in Wolfram syndrome patients identified by inactivating WFS1 mutations. Audiol Neurootol 2004;9:51-62. 12. Barjon P, Lestradet H, Labauge R. Atrophie optique primitive et surdité neurogène dans le diabète juvénile (A propos de trois observations). Presse Med 1964;72:983-986. 13. Laffay G, Lestradet H. Diabète juvénile et atrophie optique primitive. Sem Hop 1974;50:127-141. 14. Megighian D, Savastano M. Wolfram syndrome. Int J Pediatr Otorhinolaryngol 2004;68:243- 247. 15. Higashi K. Otologic findings of DIDMOAD syndrome. Am J Otol 1991;12:57-60.

135 General discussion

16. Sauer H, Chuden H, Gottesburen H, Schmitz-Valckenberg P, Seitz D. Familiäres Vorkommen von Diabetes mellitus, primärer Opticusatrophie und Innenohrschwerhörigkeit. Dtsch Med Wochenschr 1973;98:243-255. 17. Lin CH, Lee YJ, Huang CY, Shieh JW, Lin HC, Wang AM, Shih BF. Wolfram (DIDMOAD) syndrome: report of two patients. J Pediatr Endocrinol Metab 2004;17:1461-1464.

136 Chapter 6

Summary / samenvatting

Chapter 6

Summary In Chapter 2 the phenotype of DFNA8/12 is discussed. A review of this disease is given in Chapter 2.1 (in Dutch). Chapter 2.2 reports on a novel TECTA mutation, p.R1890C, found in a Dutch family with nonsyndromic autosomal dominant sensorineural hearing impairment. This presumably congenital hearing impairment occurred in the midfrequency range, amounting to about 40 dB at 1 kHz. Speech recognition was good with all phoneme recognition scores higher than 90%. An intact horizontal vestibulo-ocular reflex was found in four patients tested. The missense mutation was located in the zona pellucida domain of D-tectorin. Statistical tests on genotype-phenotype correlations of the present and collective data on DFNA8/12 revealed a significant association between the affected Į-tectorin domain (zona pellucida or zona adherens) and the type of hearing impairment (mid- or high-frequency), as well as a significant association between a cysteine-replacing substitution and age-related progression in hearing impairment. Mutations affecting the zona pellucida domain of Į-tectorin are significantly associated with midfrequency hearing impairment. Substitutions affecting amino acid residues other than cysteine show a significant association with hearing impairment without progression.

Chapter 2.3 features the results of an audiological evaluation of five selected members from this Dutch DFNA8/12(TECTA) family. In this study the audiometric characteristics were evaluated with pure tone audiometry, loudness scaling, speech perception in quiet and noise, difference limen for frequency, acoustic reflexes, oto- acoustic emissions and gap detection. Four out of five subjects showed an elevation of pure-tone thresholds, acoustic reflex thresholds, and loudness discomfort levels. Loudness growth curves were parallel to those found in normal hearing. Suprathreshold measures like difference limen for frequency modulated pure tones, gap detection, and particularly speech perception in noise were within the normal range. Distortion otoacoustic emissions were present at the higher stimulus level. These results are similar to those previously obtained from a Dutch DFNA13 family with midfrequency sensorineural hearing impairment. It seems that a defect in the tectorial membrane results primarily in an attenuation of sound, whereas suprathreshold measures, such as otoacoustic emissions and speech perception in noise, are preserved fairly well. The main effect of the defects is a shift in the operation point of the outer hair cells with near intact functioning at high levels. As most test results reflect those found in middle-ear conductive loss, in both families

139 Summary / samenvatting the sensorineural hearing impairment may be characterized as a cochlear type of conductive hearing impairment.

Chapter 3 describes the phenotype of Usher syndrome type III. In Chapter 3.1 the audiometric features, evaluated by serial pure tone audiometry and speech recognition tests, in Finnish Usher syndrome type III patients with Finmajor/Finmajor or Finmajor/Finminor USH3A mutations are presented. The group of Finnish USH3 patients demonstrated an impressively wide spectrum of progressive sensorineural hearing impairment: from normal to moderate USH2A- like hearing impairment at a young age to profound or even USH1B-like hearing impairment at a more advanced age. Compound heterozygous patients generally showed a milder phenotype. The highest progression was seen during the first two decades of life. Speech recognition started to deteriorate at highly variable ages. It is uncertain as to whether part of the hearing impairment is already present at birth or develops from early childhood onwards. Early deterioration of speech recognition may have jeopardised normal speech and language development in some patients, whereas others still had remarkably high recognition scores at a more advanced age despite considerably elevated pure tone thresholds.

Chapter 3.2 evaluates visual impairment in these Finnish Usher syndrome type III patients compared to other types of Usher syndrome (USH1B and USH2A). Cross- sectional analyses revealed significant deterioration of the functional acuity score (1.3 % per year), functional field score (1.4 % per year) and functional vision score (1.8 % per year) with advancing age in the USH3 patient group. The rate of deterioration in visual function in Finnish USH3 patients was fairly similar to that in Dutch USH1B or USH2A patients. Most of the visual function deterioration in USH3 patients occurred after deterioration in auditory function.

Chapter 4 describes the results of an international multi-centre study on hearing impairment in genotyped Wolfram syndrome patients. In all patients at least one mutation was identified in WFS1. Pure tone threshold data were used for cross- sectional analysis in subgroups of patients: male/female, young (<16 years) vs. old (19-25 years) and previously reported Dutch data vs. present additional multi-centre data. With one exception, all subgroups showed a fairly similar type of hearing impairment with, on average, thresholds of about 25 dB at 0.25-1 kHz, gently sloping downwards to about 60 dB at 8 kHz. The subgroup of previously reported Dutch

140 Chapter 6 female patients showed an exceptionally high degree of hearing impairment. With the exclusion of this subgroup, our audiometric data showed no substantial progression in hearing impairment with advancing age and no significant gender- related differences. Furthermore, it seemed that this data is representative for hearing impairment in clinically classified Wolfram syndrome patients.

141

Chapter 6

Samenvatting Dit proefschrift beschrijft klinische en genetische aspecten van DFNA8/12, het syndroom van Usher type III en het syndroom van Wolfram. Hoofdstuk 2 is gewijd aan het fenotype van DFNA8/12(TECTA) en wordt ingeleid met een review in hoofdstuk 2.1 (in het Nederlands). In hoofdstuk 2.2 wordt een nieuwe mutatie beschreven, p.R1890C, gevonden in een Nederlandse familie met een niet- syndromaal autosomaal dominant overervend gehoorverlies. Dit vermoedelijke congenitale komvormige gehoorverlies manifesteert zich in een dip van ongeveer 40 dB bij 1 kHz. Spraakverstaan was goed met foneemscores van meer dan 90%. De horizontale vestibulo-oculaire reflex was bij alle vier geteste personen intact. De missense mutatie is gelegen in het zona-pellucidadomein van D-tectorine. Analyse van genotype en fenotype toonde een significante associatie tussen het aangedane domein van Į-tectorine (zona-pellucida of zonadhesine) en het type gehoorverlies (midden- of hoogfrequent). Mutaties van het zona-pellucidadomein in het TECTA- gen veroorzaken een gehoorverlies in de middenfrequenties, terwijl mutaties in het zonadhesine domein met name een verlies in de hoge frequenties veroorzaken. Verder blijken mutaties waardoor cysteïne is vervangen door een ander aminozuur een progressief verloop van het gehoorverlies te veroorzaken.

Hoofdstuk 2.3 toont de resultaten van een uitgebreide audiologische evaluatie van vijf leden van bovengenoemde DFNA8/12(TECTA) familie. In deze studie werden de audiologische karakteristieken beoordeeld met behulp van toonaudiometrie, luidheidsschaling, spraakverstaan in stilte en ruis, het juist waarneembare verschil in frequentie, stapedius reflex drempels, oto-akoestische emissies en gat detectie. Vier van de vijf personen lieten een verhoging zien van de gehoordrempel, stapediusreflexdrempel en onaangename luidheidsdrempel. De luidheidsopbouw verliep parallel aan de curve voor normaal horenden. Bovendrempelige metingen, zoals het juist waarneembare verschil voor frequentie gemoduleerde tonen, gat- detectie en vooral spraakverstaan in ruis lagen binnen het normale bereik. Distorsie oto-akoestische emissies waren alleen aanwezig voor hoge stimuli. Deze resultaten komen overeen met die van een eerder onderzochte Nederlandse DFNA13 familie met middenfrequent gehoorverlies. Het lijkt erop dat een defect in het tectoriaal membraan vooral resulteert in een verzwakking van het geluid. Bovendrempelige metingen, zoals oto-akoestische emissies en spraakverstaan in ruis blijven relatief goed behouden. Het voornaamste effect van de afwijking in het tectoriaal membraan is het verschuiven van de drempel waarop de buitenste haarcellen

143 Summary / samenvatting werken. Omdat de testen grosso modo resultaten laten zien passend bij middenoor pathologie, kan dit sensorineurale gehoorverlies worden opgevat als een conductief cochleair gehoorverlies.

Hoofdstuk 3 belicht het fenotype van het syndroom van Usher type III (USH3). In hoofdstuk 3.1 worden de audiologische kenmerken, in de vorm van toon- en spraak audiometrie, voor Finse USH3 patiënten met Finmajor/Finmajor of Finmajor/ Finminor USH3A mutaties beschreven. De groep van Finse USH3 patiënten laat een indrukwekkende variatie van progressief sensorineuraal gehoorverlies zien; van normaal tot USH2A-achtig gehoorverlies op jonge leeftijd tot ernstig of zelfs USH1B- achtig gehoorverlies. Over het algemeen hadden de heterozygoten een milder fenotype. De progressie was het hoogst in de eerste twee decennia van het leven. Het spraakverstaan ging op zeer uiteenlopende leeftijden achteruit. Het is niet duidelijk of het gehoorverlies congenitaal is of dat dit zich later ontwikkelt. Bij sommige patiënten is de spraak- en taalontwikkeling ernstig belemmerd door de vroege achteruitgang van het spraakverstaan, terwijl bij anderen dit spraakverstaan, ondanks aanzienlijk verlaagde gehoordrempels, op latere leeftijd opvallend goed was.

Hoofdstuk 3.2 beschrijft het gezichtsvermogen van deze Finse patiënten en vergelijkt dit met andere typen van het syndroom van Usher (USH1B en USH2A). Cross-sectionele analyse toonde een significante afname van de functionele gezichtsscherpte score (1.3 %/jaar), functionele gezichtsveld score (1.4 %/jaar) en functionele visus score (1.8 %/jaar) met toegenomen leeftijd. De mate van de achteruitgang is vergelijkbaar met dat van Nederlandse USH1B en USH2A patiënten. De afname van de visus vond over het algemeen plaats nadat het gehoor was verslechterd.

Hoofdstuk 4 zet de resultaten uiteen van een internationale multi-center studie naar gehoorverlies in gegenotypeerde patiënten met het syndroom van Wolfram. Bij alle patiënten was tenminste één mutatie geïdentificeerd in WFS1. De gehoordrempels werden cross-sectioneel geanalyseerd voor verschillende subgroepen: man/vrouw, jong (<16 jaar) vs. oud (19-25 jaar) en eerder beschreven Nederlandse patiënten vs. aanvullende multi-center patiënten. Met uitzondering van één subgroep was het gehoorverlies ongeveer gelijk, met gehoordrempels van ongeveer 25 dB bij 0.25-1 kHz, die zakken tot 60 dB bij 8 kHz. De subgroep van

144 Chapter 6

Nederlandse vrouwen toonde een uitzonderlijke mate van gehoorverlies. Wanneer deze groep buiten beschouwing wordt gelaten is er geen sprake van geslachts- gerelateerde verschillen of substantiële progressie van het gehoorverlies. Het gehoorverlies van de overgebleven patiëntengroep werd vergeleken met klinisch gediagnosticeerde patiënten met het syndroom van Wolfram en bleek hiervoor representatief.

145

Dankwoord

Dankwoord Dit proefschrift was nooit tot stand gekomen zonder de begeleiding, ondersteuning en medewerking van de volgende personen.

Allereerst mijn promotor, prof. dr. C.W.R.J. Cremers. In ons eerste gesprek vertelde u over de inhoud van een in de nabije toekomst door mij te vervaardigen proefschrift. Naast de verrassing dat ik onderzoek ging doen naar voor mij onbekende syndromen en afkortingen, die schijnbaar iets met gehoorverlies te maken hadden, herinner ik mij vooral het enthousiasme waarmee u hierover sprak. Hartelijk dank voor uw begeleiding, aanmoediging en correcties die zonder enige twijfel van grote waarde zijn geweest voor het schrijven van dit proefschrift. Hiernaast was ik zonder u nooit in exotische plaatsen als Panningen, Delfgauw en Vaassen geweest.

Dr. Ronald Pennings, mijn copromotor, collega en dankbaar aanspreekpunt binnen de wondere wereld van de Otogenetica. Dit proefschrift bevat een drietal van je kindjes die vanzelfsprekend niet zonder jouw inspanning het daglicht hadden gezien.

Mijn andere copromotor, dr. Patrick Huygen. Fijn dat je deur altijd open stond en dat je tijd vrijmaakte om samen aan onze hiervoor beschreven artikelen te sleutelen.

Also on behalf of those mentioned above, I would like to thank all international co- authors, especially our Finnish colleagues Eeva-Marja Sankila end Leenamaija Kleemola, who entrusted us their valuable data.

Dr. Dirk Kunst, stafarts en vervent otogeneticus, met wie ik regelmatig het genoegen had om een familiedag in het UMCN of op locatie te organiseren. Ik heb veel gehad aan je ideeën en adviezen. Ik verwacht dat je binnen dit veld nog vele successen zult gaan boeken.

Dat laatste geldt ook voor jou, dr. Hannie Kremer, moleculair-geneticus en mijn gids/tolk in de Antropogenetica. Bedankt voor je begeleiding. Ook gaat mijn dank uit naar de medewerkers van je afdeling, met name dr. Arjan de Brouwer en dr. Rob Colin, voor al het werk dat ten grondslag ligt aan dit proefschrift.

147 Dankwoord

Dr. ir. Arjan Bosman, audioloog van het volwassen audiologisch centrum en geestelijk vader van de testbatterij beschreven in hoofdstuk 2.3. Hoog inzetten loont. Hierbij wil ik tevens “je dames” hartelijk danken voor alle werkzaamheden die hebben bijgedragen aan ons onderzoek.

Natuurlijk dank ik alle patiënten en hun familieleden, die welwillend hebben meegewerkt aan dit onderzoek. Ook wil ik al het ondersteunend personeel van onze afdeling bedanken voor de geboden hulp en in het bijzonder Diny Helsper-Peters voor het verzorgen van de lay-out.

Al mijn collega arts-assistenten Keel-, Neus- en Oorheelkunde van het UMC St. Radboud wil ik hierbij danken voor de in een enkel geval onderzoeksgerelateerde inzet, maar bovenal voor de onze afdeling zo kenmerkende gezelligheid.

Mijn paranimfen, Lukas Lisowski en Bas Jongtien. Dit boekje kwam zonder jullie hulp tot stand, maar ik hoop het samen, met de jongens, succesvol te gaan verdedigen.

Mijn familie en vrienden, van wie ik gelukkig kan zeggen dat ik niet het idee heb dat de tijd die hier ingestoken is ten koste van jullie is gegaan. Het is fantastisch jullie om me heen te hebben en om vooral over niet-medische zaken van gedachte te kunnen wisselen. Ik hoop dat jullie allemaal op 1 juni naar Nijmegen kunnen komen!

Lieve Mama, Femke en Jurre, ontzettend bedankt voor jullie onvoorwaardelijke steun (en de correcties van de Nederlandse stukken).

Tot slot, Eveline. Ik had niet durven dromen dat jij op deze plaats zou staan. Het afronden van dit laatste hoofdstuk van mijn proefschrift valt samen met het begin van een fantastisch nieuw boek dat ik met jou wil schrijven.

148 Curriculum Vitae

Curriculum Vitae Rutger Feije Plantinga is op 16 oktober 1975 geboren te Sneek. In 1994 slaagde hij daar aan de RSG Magister Alvinus voor zijn eindexamen atheneum en ging vervolgens Geneeskunde studeren aan de Universiteit Utrecht. Tijdens een coschap Keel- Neus- en Oorheelkunde in Harvard’s Massachusetts Eye and Ear Infirmary te Boston groeide zijn enthousiasme voor dit specialisme. Een keuzecoschap KNO van acht weken in het Gelderse Vallei ziekenhuis (Ede) versterkte dat beeld. In februari 2003 solliciteerde hij met goed gevolg naar een opleidingsplaats tot KNO-arts in het UMCN St Radboud. De basis voor dit promotieonderzoek is gelegd in het jaar voorafgaand aan deze opleiding, die hij naar verwachting in september 2009 zal afronden.

149

List of Publications

List of publications Plantinga RF, Frohn-Mulder IM, Takkenberg HJ, Bennink GB, Meijboom EJ, Bogers AJ. De operatieve behandeling van het hypoplastisch linkerhartsyndroom: beperkt succes. Nederlands Tijdschrift voor Geneeskunde 2001;145:85-90.

Lisowski LA, Verheijen PM, Plantinga RF, Bennink GB, Stoutenbeek P, Meijboom EJ. Het hypoplastisch linkerhartsyndroom: een studie bij 32 antenataal gediagnosticeerde casus - beleid en uitkomst. Nederlands Tijdschrift voor Perinatale Geneeskunde 2002;2:50-56.

Verheijen PM, Lisowski LA, Plantinga RF, Hitchcock JF, Bennink GB, Stoutenbeek P, Meijboom EJ. Prenatal diagnosis of the fetus with hypoplastic left heart syndrome management and outcome. Herz 2003;28:250-6. de Ru JA, Plantinga RF, Majoor MH, van Benthem PP, Slootweg PJ, Peeters PH, Hordijk GJ. Warthin's tumour and smoking. Acta Oto-Rhino-Laryngologica Belgica 2005;1:63-6.

Plantinga RF, Kleemola L, Huygen PLM, Joensuu T, Sankila E-M, Pennings RJE, Cremers CWRJ. Serial audiometry and speech recognition findings in Finnish Usher syndrome type III patients. Audiology & Neuro-Otology 2005;10:79-89.

Plantinga RF, Pennings RJE, Huygen PLM, Sankila E-M, Tuppurainen K, Kleemola L, Cremers CWRJ, Deutman AF. Visual impairment in Finnish Usher syndrome type III. Acta Ophthalmologica Scandinavica 2006;84:36-41.

Plantinga RF, de Brouwer APM, Huygen PLM, Kunst HPM, Kremer H, Cremers CWRJ. A novel TECTA mutation in a Dutch DFNA8/12 family confirms genotype-phenotype correlation. Journal of the Association for Research in Otolaryngology 2006;7:173-181.

Plantinga RF, Cremers CWRJ, Huygen PLM, Kunst HPM, Bosman AJ. Audiological evaluation of affected members from a Dutch DFNA8/12 (TECTA) family. Journal of the Association for Research in Otolaryngology 2007;8:1-7.

Cremers CWRJ, Plantinga RF, Kremer H. Van gen tot ziekte; DFNA8/12, een autosomaal dominant overervende vorm van komvormig gehoorverlies. Nederlands Tijdschrift voor Geneeskunde 2007;151:531-4.

151