LETTER TO JMG

A fifth locus for otosclerosis, OTSC5, maps to chromosome

3q2224
K Van Den Bogaert, E M R De Leenheer, W Chen, Y Lee, P Nurnberg, R J E Pennings,
K Vanderstraeten, M Thys, C W R J Cremers, R J H Smith, G Van Camp
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J Med Genet 2004;41:450453. doi: 10.1136/jmg.2004.018671
O
tosclerosis is caused by abnormal bone homeostasis of
the otic capsule leading to bony fixation of the
stapedial footplate in the oval window. Because the
transmission of sound waves from outer to inner ear is
disturbed by this fixation, the disease is characterised by
conductive hearing impairment.
1
In some cases, an additional
sensorineural component develops across all frequencies,
leading to mixed hearing impairment.
2 3
The conductive
component of the hearing impairment can be restored by
stapes replacing microsurgery; however, the sensorineural
component cannot surgically be corrected.
4
Otosclerosis has a prevalence of 0.30.4% in the Caucasian
population.
5
The etiology of the disease is unknown, but
epidemiological studies indicate the involvement of genetic
as well as environmental factors. However, large families
segregating otosclerosis are very rare, whereas there are
frequent sporadic cases and smaller families with only a few
affected members. Based on these findings, otosclerosis can
be considered a genetically complex disease, caused by an
interaction of genes and environmental factors, but with rare
monogenic forms.
To date, three autosomal dominant otosclerosis loci have
been reported: OTSC1 on chromosome 15q2526,
6
OTSC2 on
chromosome 7q3436,
7
and OTSC3 on chromosome 6p2122.
8
In addition, a fourth locus, OTSC4, has been reserved by the
Human Genome Organisation nomenclature committee, but
this has not been published. None of the corresponding genes
have been cloned. In this study, we identified a large Dutch
family segregating an autosomal dominant otosclerosis. After
exclusion of the known loci, a genome-wide screen and
linkage analysis in this family revealed the existence of a
fifth otosclerosis locus, OTSC5, localised on chromosome
3q2224.
METHODS
Cl i ni cal di agnosi s
The family was identified via the Department of
Otorhinolaryngology of the University Medical Center
St.-Radboud Nijmegen (The Netherlands) (fig 1). Pure-tone
audiometry was performed in all persons with air conduction
at 125, 250, 500, 1000, 2000, 4000, and 8000 Hz, and bone
conduction at 250, 500, 1000, 2000, and 4000 Hz. Tympanic
membrane compliance and ipsi- and contralateral stapedial
reflex decay were also measured. Individuals in whom stapes
fixation with an otosclerotic focus was confirmed during
stapes replacing microsurgery were considered affected. In
non-operated persons, the clinical diagnosis of otosclerosis
was based on audiologic data. Persons with a conductive or
mixed hearing loss together with absent or immeasurable
stapedial reflexes were classified as affected. Due to the
variability of the age of onset, only family members .50
years of age and with normal hearing were considered
unaffected. Information on deceased members of the
pedigree was obtained by history.
Genotype anal ysi s
Blood samples from study participants were obtained after
informed consent and were used as a source of genomic DNA,
which was isolated using standard techniques. The micro-
satellite markers used to analyse linkage to the known
otosclerosis loci were D15S652, D15S1004, and D15S657 for
OTSC1, D7S495, D7S2560, D7S684, D7S2513, and D7S2426
for OTSC2, and D6S1588, D6S273, D6S291, D6S1680, and
D6S426 for OTSC3. Information for all markers was taken
from The Genome Database (http://www.gdb.org/).
A genome-wide screen was performed by fluorescence-
based semi-automated genotyping using 380 microsatellite
markers selected from the final Ge nethon linkage map
9
with
an average heterozygosity of 0.76. For fine mapping of the
candidate region additional microsatellite markers were
selected from public databases. After individual PCR ampli-
fication on an MJ Research thermocycler, PCR products were
pooled and size fractionated by electrophoresis on a Prism
ABI 3700 DNA sequencer (Applied Biosystems). Allele sizes
were determined using GENESCAN 2.1.1. and Genotyper
V3.7 software (Applied Biosystems).
Key poi nts
N
Otosclerosis, a bone disorder of the otic capsule, is the
most common cause of hearing impairment among
white adults with a prevalence of 0.30.4%. The
etiology of the disease is unknown, but there are
strong suggestions that genetic as well as environ-
mental factors may be involved.
N
To date, three autosomal dominant otosclerosis loci
(OTSC1 on chromosome 15q2526, OTSC2 on
chromosome 7q3436, and OTSC3 on chromosome
6p2122) have been reported, while a fourth locus,
OTSC4, has been reserved. None of the responsible
genes have been cloned.
N
In this study, a fifth locus, OTSC5, was localised on
chromosome 3q2224 by linkage analysis in a Dutch
pedigree segregating autosomal dominant otosclero-
sis. Recombinant individuals delineate a 15.5 Mb
candidate interval between the markers D3S1292
centromeric and D3S1744 telomeric.
N
In the future, refinement of the candidate region and
mutation analysis of candidate genes will lead to the
identification of the disease-causing gene in this family.
450
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Li nkage anal ysi s
All genotypes were checked for Mendelian inconsistencies
using LINKRUN software (Wienker, unpublished). Two-point
LOD scores were calculated between each marker locus
and otosclerosis under the assumption of an autosomal
dominant model with 90% penetrance, a phenocopy rate of
1%, a frequency of 0.0001 for the disease allele, and equal
allele frequencies for each marker using LINKAGE V5.21
software.
10
Equal recombination frequencies between males
and females were assumed.
Mutati on anal ysi s
Primers were designed from intronic sequences flanking the
exons of PCOLCE2 and CHST2 to amplify 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 Dyenamic
TM
ET
Terminator Cycle Sequencing Kit (Amersham Biosciences).
RESULTS AND DI SCUSSI ON
Cl i ni cal anal ysi s
The family presented in this study segregates an autosomal
dominant otosclerosis (fig 1) in eight of 10 affected members
confirmed by stapes replacing microsurgery. In the two
remaining subjects (III:11 and III:12), the presence of an air
bone gap, closing at a frequency of 2000 Hz, together with
the absence of stapedial reflexes clinically confirmed the
diagnosis of otosclerosis. Nine family members and one
spouse had normal hearing.
Geneti c anal ysi s
Initially, genetic analysis was performed on 18 family
members, of whom 10 were affected and seven unaffected,
with one unaffected spouse (fig 1). Statistically significant
exclusion of the three known otosclerosis loci was demon-
strated (table 1). A genome-wide screen in this family
revealed suggestive linkage at chromosome region 3q2224,
from which a maximum two-point LOD score of 2.98 was
obtained for marker D3S1569 at h =0.05. Two additional
unaffected subjects were identified (III:14 and III:18). After
genotyping these individuals for markers in the region of
interest, a maximum two-point LOD score of 3.46 was
obtained for marker D3S1569 at h =0.05 (table 2).
Haplotypes were constructed to confine the interval of the
linked region. A recombination event in individuals III:5 and
III:9 placed the disease locus proximal to D3S1744, while a
recombination event in individuals III:12 and III:13 mapped
the disease-causing gene distal to D3S1292. Hence, the
maximal interval of linkage with the otosclerosis pheno-
type is bordered by D3S1292 (centromeric) and D3S1744
(telomeric) within a region of ,15.5 Mb, according to the
last draft of the human genome sequence (Build 34 Version
1). A physical map of the candidate region is presented in
figure 2.
Individual III:2, who in the linkage analysis was consid-
ered to be affected, does not carry the haplotype segregating
with the disease in the other patients. Because he underwent
bilateral stapes replacing microsurgery, he could be consid-
ered to be a phenocopy. In view of the high frequency of
otosclerosis in the white population, the presence of a
phenocopy in an otosclerosis family is not surprising.
Alternatively, this could reflect an unrecognised double
crossover, which is extremely unlikely, given the small
intermarker distances.
The 15.5 Mb OTSC5 interval contains 59 identified genes
and 45 gene predictions. Two genes were considered very
good otosclerosis candidate genes: PCOLCE2 and CHST2. The
PCOLCE2 (procollagen COOH-terminal proteinase enhancer
protein 2) gene product is found to be a glycoprotein that
binds the COOH-terminal propeptide of type I procollagen
and is highly expressed in non-ossified cartilage in develop-
ing tissues.
11
The otic capsule is unique in retaining calcified
I:1
(2)
(1)
(2)
(1)
(2)
(2)
(2)
(2)
(4)
(?)
(?)
(?)
I:2
(2)
(4)
(1)
(2)
(1)
(3)
(2)
(3)
(?)
(?)
(?)
(?)
II:1
(3)
(5)
(1)
(1)
(3)
(3)
D3S1558
D3S1303
D3S1292
D3S3554
D3S1569
D3S1744
(3)
(2)
(1)
(1)
(2)
(4)
II:2
(2)
(1)
(2)
(1)
(2)
(2)
(2)
(4)
(1)
(2)
(1)
(3)
II:3
(2)
(2)
(4)
(1)
(2)
(2)
(2)
(3)
(1)
(2)
(1)
(3)
II:4
(3)
(3)
(2)
(1)
(3)
(1)
(3)
(1)
(1)
(3)
(6)
(2)
III:6
2
3
3
3
5
1
3
5
3
1
4
5
III:8
3
5
1
1
3
3
2
4
1
2
1
3
III:9
3
2
1
1
3
3
2
4
1
2
1
2
III:11
3
5
1
1
2
3
2
1
2
1
2
2
III:15
2
2
1
2
1
3
3
3
2
1
3
1
III:18
2
2
4
2
1
3
3
1
1
3
6
2
III:1
3
5
1
1
3
3
2
1
2
1
2
2
2
2
4
1
2
2
3
1
1
3
6
2
III:7
3
5
1
1
3
3
2
1
2
1
2
2
III:3 III:4
3
5
1
1
3
3
2
4
2
1
2
2
III:2
3
5
1
1
3
3
2
4
1
2
1
3
III:5
3
2
1
1
2
4
2
4
1
2
1
2
III:10
3
5
1
1
3
3
2
1
2
1
2
2
III:12
2
2
4
1
2
2
3
3
2
1
3
1
III:13 III:14
2
3
1
2
1
3
3
3
1
3
6
2
III:16
2
3
1
2
1
3
3
1
1
3
6
2
III:17
2
3
1
2
1
3
3
3
2
1
3
1
IV:1
1
4
1
3
4
6
2
4
2
1
3
3
IV:2
2
3
3
3
5
1
2
1
2
1
2
2
IV:3
3
5
3
1
4
5
3
5
2
1
2
2
Figure 1 Pedigree of the Dutch family with autosomal dominant otosclerosis, showing the most likely haplotypes for the chromosome 3 markers. The
haplotype linked to otosclerosis is indicated with a black bar.
Letter to JMG 451
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cartilage, known as globuli interossei, throughout life and in
lacking bone remodelling. Because these factors are unique to
the otic capsule they may predispose to otosclerosis. The
expression pattern of PCOLCE2 suggests a possible role for
this gene in the pathogenesis. The CHST2 (carbohydrate
sulfotransferase 2) gene product is a Golgi-associated sulfo-
transferase. These sulfotransferases sulfonate glycoproteins,
glycosaminoglycans, peptidyl tyrosine, and heparan sulfates
and play important roles in intercellular communication.
12
Because otosclerosis has been hypothesised to result from a
disorder of the extracellular matrix of the cartilaginous rests
present in the adult temporal bone, the clear relationship of
CHST2 to extracellular matrix function makes this gene a
strong candidate gene. However, mutation analysis of the
Figure 2 Localisation of OTSC5 on chromosome 3q2224. The black bar indicates the region that contains the gene for otosclerosis.
Table 1 Two-point LOD scores between otosclerosis and the known otosclerosis loci
OTSC1, OTSC2, and OTSC3
Locus Marker
Recombination fraction (h)
0.0 0.01 0.05 0.1 0.2 0.3 0.4
OTSC1 D15S652 26.67 24.68 22.88 21.88 20.80 20.27 20.04
D15S1004 25.57 24.57 22.83 21.83 20.81 20.31 20.07
D15S657 20.03 20.01 0.05 0.09 0.13 0.12 0.07
OTSC2 D7S495 25.57 23.91 22.56 21.72 20.76 20.29 20.06
D7S2560 23.91 22.19 20.93 20.31 0.19 0.28 0.13
D7S684 21.48 20.13 0.46 0.64 0.63 0.42 0.14
D7S2513 24.36 22.86 21.84 21.19 20.44 20.10 0.00
D7S2426 24.04 22.51 21.40 20.87 20.38 20.15 20.04
OTSC3 D6S1588 20.71 20.63 20.37 20.18 0.00 0.04 0.02
D6S273 21.06 20.99 20.75 20.50 20.18 20.05 20.02
D6S291 22.41 22.07 21.41 20.99 20.58 20.33 20.13
D6S1680 22.70 22.31 21.54 21.05 20.51 20.21 20.05
D6S426 24.10 23.48 22.30 21.50 20.63 20.22 20.05
452 Letter to JMG
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coding region and the intronexon boundaries of both genes
did not reveal any disease-causing mutation.
Identification of new otosclerosis loci will increase the
possibility of detecting the first otosclerosis-causing gene in
the near future. Identification of this first gene will probably
facilitate the identification of the causative genes localised in
the other known otosclerosis loci. Characterisation of these
genes will give us increased knowledge regarding the
underlying molecular pathway of otosclerosis and of the otic
capsule in general.
ACKNOWLEDGEMENTS
We thank the family members for their cooperation, and Inka
Szangolies, Regina Pospiech, and Gudrun Nurnberg for excellent
technical support. KVDB holds a predoctoral research position with
the Instituut voor de aanmoediging van Innovatie door Wetenschap
en Technologie in Vlaanderen (IWT-Vlaanderen). This research was
performed in the framework of the Interuniversity Attraction Poles
program P5/19 of the Belgian Federal Science Policy Office.
Authors affiliations
. . . . . . . . . . . . . . . . . . . . .
K Van Den Bogaert, K Vanderstraeten, M Thys, G Van Camp,
Department of Medical Genetics, University of Antwerp,
Universiteitsplein 1, 2610 Antwerp, Belgium
E M R De Leenheer, R J E Pennings, C W R J Cremers, Department of
Otorhinolaryngology, University Medical Center St Radboud, Philips van
Leydenlaan 15, 6500 HB Nijmegen, The Netherlands
E M R De Leenheer, Department of Otorhinolaryngology and Head and
Neck Surgery, University Hospital Ghent, De Pintelaan 185, 9000
Ghent, Belgium
W Chen, R J H Smith, Molecular Otolaryngology Research Laboratories,
Department of Otolaryngology, University of Iowa, 200 Hawkins Drive,
Iowa City, IA 52242, USA
Y Lee, P Nurnberg, Gene Mapping Center (GMC), Max Delbrueck
Center for Molecular Medicine (MDC) Berlin-Buch, Robert-Roessle-
Strasse 10, D-13092 Berlin, Germany
This study was supported in part by NIH grant R01DC05218 (RJHS,
GVC), and grants from the University of Antwerp (GVC) and from the
Vlaams Fonds voor Wetenschappelijk Onderzoek (FWO) (GVC).
Conflict of interest: none declared.
Correspondence to: Guy Van Camp, Department of Medical Genetics,
University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium;
Guy.VanCamp@ua.ac.be
Received 21 January 2004
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Table 2 Two-point LOD scores between otosclerosis and chromosome 3 markers
(OTSC5)
Marker
Recombination fraction (h)
0.0 0.01 0.05 0.1 0.2 0.3 0.4
D3S1558 21.23 20.96 20.54 20.32 20.13 20.05 20.01
D3S1303 23.74 22.24 20.98 20.38 0.09 0.17 0.08
D3S1292 21.29 20.25 0.72 1.05 1.07 0.75 0.29
D3S3554 2.18 2.31 2.43 2.31 1.83 1.18 0.45
D3S1569 3.13 3.33 3.46 3.28 2.64 1.78 0.78
D3S1744 1.14 1.42 1.82 1.93 1.71 1.20 0.53
Letter to JMG 453
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