Monogenic nonsyndromic otosclerosis: Audiological and linkage analysis in a large Greek pedigree

International Journal of Pediatric Otorhinolaryngology (2006) 70, 631—637

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Monogenic nonsyndromic otosclerosis: Audiological and linkage analysis in a large Greek pedigree Vassiliki Iliadou a,1, Kris Van Den Bogaert b,1, Nikolaos Eleftheriades a, George Aperis c, Kathleen Vanderstraeten b, Erik Fransen b, Melissa Thys b, Maria Grigoriadou c, Andreas Pampanos c, John Economides d, Theophilos Iliades a, Guy Van Camp b, Michael B. Petersen c,* a

Audiology Unit, Department of Otorhinolaryngology, Aristotle University of Thessaloniki, AHEPA Hospital, GR-54006 Thessaloniki, Greece b Department of Medical Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium c Department of Genetics, Institute of Child Health, ‘‘Aghia Sophia’’ Children’s Hospital, GR-11527 Athens, Greece d Department of Audiology-Neurootology, ‘‘Aghia Sophia’’ Children’s Hospital, GR-11527 Athens, Greece Received 2 April 2005; accepted 12 August 2005

KEYWORDS Audiological analysis; Genetic linkage analysis; Monogenic inheritance; Otosclerosis

Summary Objective: The aim of our study was to characterize the hearing impairment in a large multigenerational Greek family with autosomal dominant nonsyndromic otosclerosis and to perform genetic linkage analysis to known otosclerosis loci and collagen genes. In addition, we looked for mutations in the NOG gene to rule out congenital stapes ankylosis syndrome. Methods: Audiological analysis of the affected persons was based on multiple linear regression (MLR) analysis and construction of age-related typical audiograms (ARTA). Genotyping of microsatellite DNA polymorphisms for known otosclerosis (OTSC) loci or collagen genes and linkage analysis using the MLINK computer program were performed. The coding region of the NOG gene was screened for mutations by direct DNA sequencing. Results: The hearing loss in this family appears in childhood as conductive, but soon becomes mixed. Because the additional sensorineural component is progressive, this finally has lead to a pure sensorineural hearing loss in some family members, as the conductive component is masked. Audiological analysis showed an age-independent

* Corresponding author. Tel.: +30 210 7467789; fax: +30 210 7700111. E-mail address: [email protected] (M.B. Petersen). 1 The first two authors contributed equally to this work. 0165-5876/$ — see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2005.08.012

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conductive component and a progressive frequency-specific sensorineural component. Linkage analysis excluded linkage to the four known otosclerosis loci (OTSC1, OTSC2, OTSC3, and OTSC5), as well as to the COL1A1 and COL1A2 genes. Mutation analysis of the coding region of the NOG gene did not reveal any disease causing mutation. Conclusions: This study represents the first description of a detailed audiological analysis in a large pedigree segregating otosclerosis as a monogenic autosomal dominant trait. Exclusion of the four known otosclerosis loci in this family shows that monogenic otosclerosis is a genetically heterogeneous disease involving at least five different genes. A mutation in the NOG gene is not the underlying molecular mechanism of the early onset otosclerosis segregating in this family. # 2005 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Otosclerosis of the oval window (stapedio-vestibular joint), which interferes with the motion of the stapes, has a prevalence of about 0.3—0.4% among white adults and manifests as a conductive hearing loss (CHL) [1,2]. It is estimated that in about 10% of all otosclerosis cases, the otosclerotic foci encroach on the labyrinthine capsule, producing an additional sensorineural hearing loss (SHL) [3,4]. Otosclerotic damage to the cochlea, as shown by temporal bone histological specimens, can co-exist with oval window ankylosis, but it can also occur without stapes fixation [5]. In this latter case, the cause of the SHL is difficult to trace by a routine audiometrical investigation, when the HL is the only symptom and no other predisposing external factor can be recognized. From a genetic point of view, otosclerosis is usually considered as a multifactorial disease. About 50% of patients lack a positive family history, and if there is one, the family is rarely large enough for genetic linkage studies. The few families described with monogenic inheritance of otosclerosis, have shown autosomal dominant inheritance often with reduced penetrance. Four autosomal dominant nonsyndromic otosclerosis loci have so far been published: OTSC1 on chromosome 15q25—q26 [6], OTSC2 on chromosome 7q34—q36 [7], OTSC3 on chromosome 6p21.3—p22.3 [8], and OTSC5 on chromosome 3q22—q24 [9]. The name OTSC4 has been reserved with the Human Genome Nomenclature committee, but this locus has not been published yet. None of the genes responsible for these forms of otosclerosis with monogenic inheritance, have been identified so far. In contrast to the rarity of large families with monogenic inheritance of otosclerosis, sporadic cases of otosclerosis are very common. Such patients can be used in case-control studies to search for susceptibility genes involved in the multifactorial forms of otosclerosis. The only suggestion thus far of an otosclerosis susceptibility gene originates from an association study between the

COL1A1, COL1A2, and COL2A1 genes and otosclerosis [10]. The COL1A1 and COL1A2 genes were selected because of the striking clinical and histopathological similarities between otosclerosis and osteogenesis imperfecta, caused by mutations in these two collagen genes [11]. On the other hand, the COL2A1 gene is abundantly expressed in the otic capsule [12,13]. A statistically significant association was detected between the COL1A1 gene and otosclerosis, while no association could be found with the COL1A2 and COL2A1 genes [10]. The large Greek family with monogenic, autosomal dominant otosclerosis presented in this study, underwent a detailed clinical analysis based on a consecutive statistical audiological analysis. Agerelated typical audiograms (ARTA) were constructed, visualizing the progression of the hearing impairment in this family. Furthermore, we analyzed this family for genetic linkage to the four known otosclerosis loci, as well as for linkage to the COL1A1 and COL1A2 genes. In addition, given the early age of onset, we looked for mutations in the NOG gene. Mutations in NOG, the gene encoding noggin, have been identified in three stapes ankylosis syndromes, namely proximal symphalangism, multiple synostoses syndrome, and stapes ankylosis with broad thumb and toes [14,15]. A congenital stapes ankylosis syndrome may be difficult to differentiate from otosclerosis when associated skeletal anomalies are subtle, such that a syndrome is not recognized.

2. Material and methods 2.1. Family recruitment In this study, we present a large multigenerational Greek family, the members of which originate from a small village in the island of Rhodes (Fig. 1). Special attention is drawn to a consanguineous couple (individuals V:3 and V:4), with both of them having HL.

Monogenic nonsyndromic otosclerosis

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Fig. 1 Pedigree of the Greek family with autosomal dominant inheritance of otosclerosis. Family members, whose DNA was analyzed, are indicated with an asterisk. Affected family members are represented by solid symbols, male family members by squares, and female members by circles. Family members that were not clinically investigated or with an atypical hearing impairment are indicated with a question mark.

2.2. Clinical analysis After informed consent, we examined 46 members of this family. A detailed questionnaire was filled out to exclude patients with chronic middle ear disease, noise induced HL or other factors that would categorize the member as being at high risk for acquired HL. Anamnestic information was also obtained as to previous audiological assessment, age of onset of the HL if present, use of hearing aids, and progression of HL. All family members with HL were also examined by a clinical geneticist to exclude syndromic forms of otosclerosis. In particular, movement of finger joints, shape of fingers and toes as well as facial characteristics were noted. Otoscopy was performed to rule out outer ear pathology, while tympanometry was conducted to exclude middle ear pathology. Stapedial reflexes were obtained to assess the motion of the stapes. Tuning fork tests (Rinne and Bing) discriminated the type of HL as either CHL or SHL. Pure tone audiometry was performed, for air conduction at frequencies 250, 500, 1000, 2000, 4000, and 8000 Hz, and for bone conduction at frequencies 250, 500, 1000, 2000, and 4000 Hz.

2.3. Audiological analysis The air conduction, bone conduction and air-bone gap threshold values were plotted versus age for

each frequency separately. Progression of the hearing impairment was analyzed at each frequency by multiple linear regression (MLR) analysis, with age and gender as the independent variables and threshold as outcome variable. Hence, three regression equations were obtained for each frequency: one for air conduction, one for bone conduction and one for the air-bone gap. For each of these equations, the regression coefficient for age represents the frequency-specific annual threshold deterioration (ATD) expressed in dB/year. The regression equations were used to construct age-related typical audiograms at 10, 20, 30, 40, 50, and 60 years of age [16].

2.4. Genotyping Blood samples were obtained after informed consent (in accordance with the ethical standards of the Ethical Committee of the Institute of Child Health, Athens) and used as a source of isolation of genomic DNA using standard techniques. Three microsatellite DNA polymorphisms for each OTSC locus or collagen gene were analyzed (Table 1). Polymerase chain reaction (PCR) amplification was carried out using standard conditions. One of the primers was synthesized with an M13 sequence at the 50 end. A fluorescently labeled M13 primer was included in the PCR reaction, thus labeling the PCR product. Capillary electrophoresis and pattern visualization

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Table 1 Two-point LOD scores between the otosclerosis phenotype and microsatellite markers for the OTSC1, OTSC2, OTSC3, OTSC5, COL1A1, and COL1A2 loci Locus

Markers

Recombination frequency 0.00

0.01

0.05

0.1

0.2

0.3

0.4

OTSC1

D15S652 D15S1004 D15S657


2.50
6.07
6.48


2.15
5.23
5.26


0.88
3.30
2.89


0.16
2.12
1.63

0.26
0.96
0.58

0.24
0.42
0.18

0.10
0.15
0.03

OTSC2

D7S495 D7S2513 D7S2426


7.29
7.42
7.87


5.05
5.87
4.98


2.76
3.57
2.76


1.66
2.28
1.63


0.64
1.03
0.66


0.19
0.44
0.25


0.01
0.14
0.06

OTSC3

D6S1660 D6S291 D6S1680


8.89
2.81
2.20


5.37
2.23
1.86


2.87
0.97
0.90


1.73
0.43
0.35


0.71
0.02 0.09


0.26 0.08 0.17


0.06 0.08 0.09

OTSC5

D3S1292 D3S3694 D3S1744


6.08
7.57
4.08


4.79
5.06
2.74


2.74
2.77
1.34


1.60
1.70
0.72


0.58
0.76
0.25


0.16
0.32
0.09


0.01
0.09
0.02

COL1A1

D17S797 D17S941 D17S788


6.71
5.50
5.27


3.25
3.72
4.09


1.91
2.12
2.23


1.36
1.30
1.28


0.73
0.54
0.49


0.30
0.20
0.19


0.08
0.05
0.08

COL1A2

D7S644 D7S2430 D7S651


4.57
9.42
4.41


3.65
6.02
2.32


2.43
3.24
0.71


1.66
2.03
0.05


0.83
1.05 0.35


0.39
0.63 0.30


0.14
0.31 0.12

were performed using an ABI PRISM 3100 (Applied Biosystems Inc.).

3. Results 3.1. Clinical analysis

2.5. Linkage analysis Two-point LOD scores were calculated using the MLINK computer program [17]. The linkage parameters were chosen in compliance with older studies, suggesting that otosclerosis is inherited as an autosomal dominant trait with reduced penetrance [18—22]. As standard linkage parameters, the frequency of the otosclerosis gene was set at 0.0001 and the disease was assumed to be 90% penetrant and autosomal dominant. To allow for possible phenocopies, this chance was set at 1%, because without surgical exploration it is often difficult to exclude HL of other reasons. Equal recombination frequencies between males and females were assumed. For each marker, the number of alleles in the LOD score calculations was set at the observed number (N) of alleles in the pedigree, and allele frequencies were set at 1/N.

2.6. Mutation analysis The NOG coding region was amplified from genomic DNA from patient and control individuals. Direct sequencing of the PCR product was performed on both forward and reverse strands on an ABI 3100 sequencer using the DyenamicTM ET Terminator Cycle Sequencing Kit (Amersham Biosciences).

The family presented in this study segregates an autosomal dominant nonsyndromic, progressive HL (Fig. 1). The HL in this family appears in childhood at the age of about 10 years as a CHL, but soon becomes mixed (MHL). In some of the family members, progression of the additional sensorineural component finally leads to a pure SHL, when the sensorineural component becomes greater than the conductive. Twenty-four family members were diagnosed as having HL. Fifteen persons showed a clear conductive component in one or both ears producing a CHL or, when an additional sensorineural component was present, an MHL. Nine patients presented a pure SHL. The presence of an air-bone gap, closing at a frequency of 2000 Hz, in the persons with a CHL or MHL together with the absence of stapedial reflexes in all patients with HL, clinically confirmed the diagnosis of otosclerosis in these individuals. Individual VII:3 showed an atypical hearing impairment and was excluded from further calculations. An MRI scan of this child showed a large vestibular aqueduct with substantial dilatation of the ductus endolymphaticus and saccus endolymphaticus, and an anomaly of the diaphragms of the cochlea with imperfect separation of the top and middle turns. Twenty-two persons either had normal hearing or HL most likely due to noise or presbyacusis.

Monogenic nonsyndromic otosclerosis

3.2. Audiological analysis In all calculations we worked with mean air, mean bone and mean air-bone gap thresholds. Visual inspection of the constructed scatterplots (mean air, bone and gap threshold versus age for each frequency) revealed one outlier, corresponding to individual IV:5 in the pedigree (data not shown). Because this person, an 80-year-old man, had a minimal sensorineural component and would be one of the best-hearing affected individuals, he was excluded from further calculations. An MLR analysis for each frequency was carried out to investigate the progression of air and bone conduction thresholds as well as the progression of the air-bone gap. First of all, this analysis revealed no effect of gender on the progression rate for any of the frequencies, enabling us to pool males and females in further calculations. The air-bone gap, representing the conductive component of the hearing loss due to stapes fixation, seemed to be independent of age within each frequency. However, a one-way ANOVA analysis revealed that this gap is not constant across different frequencies.

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Concerning the air-bone gap in this family, we can thus conclude that the conductive component of the hearing loss is age-independent, but frequency-specific. The bone conduction loss, which measures the cochlear component of the HL, is progressive at all frequencies, but the progression rate, the ATD (dB/ year), is more prominent at the higher frequencies compared to the lower. Based upon regression equations for each frequency we constructed a bone conduction ARTA (Fig. 2A). With regard to the bone conduction in this family, we can conclude that the sensorineural component of the HL is progressive with age across all frequencies, but the progression rate is frequency-specific. The thresholds of air conduction represent the HL due to the conductive as well as the sensorineural component. This means that in this family the air conduction loss will be the sum of an age-independent air-bone gap (conductive component) and an age-dependent bone conduction loss (sensorineural component). Consequently, the air conduction loss will be progressive, because it results from the sum of a constant factor and a progressive factor, and the

Fig. 2 Presentation of bone conduction (A) and air conduction (B) ARTAs, representing the progression of the HL per decade, characteristic of the Greek otosclerosis pedigree reported.

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ATDs will be the same as those for the bone conduction loss. However, the construction of air conduction ARTAs reveals that the shape of the curves differs from the bone conduction ARTAs, due to the fact that the air-bone gap is constant, but frequency-specific (Fig. 2B).

3.3. Linkage analysis Genetic analysis was performed on a subset of 31 family members, including 16 affected members, 7 unaffected members, 5 spouses and 3 family members (individuals IV:5, IV:7 and VII:3) with an uncertain diagnosis (Fig. 1). The spouses and individual IV:7 did not undergo an audiometrical examination, while persons IV:5 and VII:3 showed an atypical hearing impairment, as described in the clinical and audiometrical analysis sections. Haplotypes were constructed (data not shown) and two-point LOD scores were calculated. Statistically significant exclusion of the four known otosclerosis loci was demonstrated (Table 1), indicating that an additional, as yet unknown otosclerosis locus must exist. Exclusion of linkage to the COL1A1 and COL1A2 genes shows that mutations in these genes are not the underlying molecular mechanism of the pathology transmitted in this pedigree (Table 1).

3.4. Mutation analysis Given the early age of onset in this family, we looked for mutations in the NOG gene by DNA sequencing to rule out a congenital stapes ankylosis syndrome. However, mutation analysis of the coding region did not reveal any disease causing mutation.

4. Discussion According to reported histological studies [23—25], three categories of otosclerosis can be distinguished: the first category is the classical one with fixation of the stapes footplate, the second one manifests as cochlear damage with concomitant oval window ankylosis and a third one as cochlear damage without stapes fixation. Investigators agree that many cases of this second and third category may remain undiagnosed. Audiological analysis of patients belonging to the second category may result in a pure SHL, even when stapes fixation, based on absence of stapedial reflexes, is detected. This can be explained by the fact that a progressive sensorineural component can dominate a smaller conductive component when audiometry is performed. Patients with a pure progressive SHL and with stapedial reflexes (third category) should be

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suspected of having otosclerosis when there is coexistence of the following clinical signs: history of a slowly progressive bilateral and often asymmetrical HL, appearance of the phenomenon of paracusis of Willis (better hearing in noisy environments), and detailed history revealing no other cause of HL, either hereditary or acquired [26]. The nonsyndromic autosomal dominant otosclerosis reported in this Greek pedigree initially involves the oval window, disturbing the free motion of the stapes footplate, thus producing a conductive HL. The otosclerosis then most likely proceeds to the labyrinthine capsule producing a mixed HL, due to the development of an additional sensorineural component. Progression of this sensorineural component finally leads to a pure SHL in some family members. The diagnosis of otosclerosis in these latter patients would have been practically impossible without the entire family pedigree. The family history together with the presence of the additional signs mentioned earlier, clinically confirmed the diagnosis of otosclerosis in these patients. MLR analysis on the audiograms of all patients revealed the presence of an age-independent, frequency-specific conductive component and a progressive, frequency-specific sensorineural component in this family. This audiometrical analysis showed that individual IV:5 has an atypical audiogram compared to the rest of the patients. Although this 80-year-old man is the oldest person analyzed, he is one of the best-hearing patients in this family. The otosclerosis of this person only consists of the constant conductive component, confirmed by the absence of stapedial reflexes. The minimal sensorineural component of his hearing loss is probably due to presbyacusis, while in nearly all the other affected family members, except for a few very young patients, a remarkable SHL is present. This profile is in sharp contrast with the general finding of a progressive sensorineural component in this family. For these reasons, this individual had been excluded from further audiometrical analyses and received an uncertain diagnosis in the genetic linkage analysis. Genetic linkage analysis of this newly ascertained large Greek otosclerosis pedigree indicated that, besides the four known otosclerosis loci, at least one additional nonsyndromic locus must exist. A future genome wide screen should elucidate the localization of the disease causing gene in this family. Although no otosclerosis causing genes have been identified so far, mapping of additional loci will make a great contribution towards the unraveling of the etiology of otosclerosis. Once the first otosclerosis causing gene will be identified, this will help us to trace additional genes causing this type of HL. Genes with a comparable function and located in one of

Monogenic nonsyndromic otosclerosis

the known candidate regions will be very attractive candidates to analyze. Functional analysis of otosclerosis causing genes will give us more information about the pathways involved in the pathogenesis.This will be an excellent starting point for the development of better therapies and/or prevention strategies, in order to reduce the prevalence of this common cause of HL among white adults.

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[9]

[10]

[11]

Acknowledgements We thank all the family members for their cooperation. This study was supported in part by NIH grant R01DC05218, grants from the University of Antwerp and from the Vlaams Fonds voor Wetenschappelijk Onderzoek (FWO) to GVC, and a grant from Oticon Fonden, Denmark, to MBP. This research was performed in the framework of the Interuniversity Attraction Poles programme P5/19 of the Federal Office for Scientific, Technical, and Cultural Affairs, Belgium.

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