Two Novel Missense Mutations in the TECTA Gene in Korean Families with Autosomal Dominant Nonsyndromic Hearing Loss
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380 Annals of Clinical & Laboratory Science, vol. 40, no. 4, 2010
Two Novel Missense Mutations in the TECTA Gene in Korean
Families with Autosomal Dominant Nonsyndromic Hearing Loss
Borum Sagong,1* Raekil Park,2* Yee Hyuk Kim,3 Kyu-Yup Lee,3 Jeong-In Baek,1 Hyun-Joo Cho,1
In-Jee Cho,3 Un-Kyung Kim,1 and Sang-Heun Lee3
1Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu;
2Vestibulocochlear Research Center, College of Medicine, Wongwang University, Iksan; and
3Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Kyungpook National
University, Daegu, South Korea
Abstract. The TECTA gene, which encodes alpha-tectorin, is known as a causative gene for DFNA8/
DFNA12, and DFNB21 hearing loss in humans. In the present study, mutation analysis of the TECTA
gene was performed in 62 Korean patients with hereditary hearing loss. Two novel nucleotide substitutions,
p.V317E and p.T1866M, were identified for the first time in the Korean population. These mutations
result in the substitution of amino acids in the zonadhesin (ZA) and the zona pellucida (ZP) domains, and
show a genotype-phenotype correlation, which is a characteristic of TECTA-related mutations in autosomal
dominant nonsyndromic hearing loss. Both mutations are located in highly conserved regions of alpha-
tectorin and were not found in 120 unrelated control subjects with normal hearing. Based on this evidence,
it is likely that both mutations are the pathogenic ones causing the hearing loss. This study provides useful
information for the functional study of hereditary hearing loss caused by tectorial membrane defects.
Keywords: hereditary hearing loss, TECTA gene, tectorial membrane, alpha-tectorin
Introduction cause autosomal dominant (DFNA8/DFNA12)
and autosomal recessive (DFNB21) nonsyndromic
The tectorial membrane of the organ of Corti is a hearing loss [7,9] and 18 different mutations have
sheet of extracellular matrix composed of three been identified [5,7,9-20]. Interestingly, all of the
different types of collagen and three noncollagenous missense mutations in the TECTA gene are
glycoproteins that are expressed at high levels only responsible for autosomal dominant nonsyndromic
in the inner ear [1]. Alpha-tectorin, a major non- hearing loss (ADNSHL), whereas the truncated
collagenous component of the tectorial membrane, mutations, including nonsense, frameshift, and
has several functional domains: the entactin (ENT) splicing, cause autosomal recessive nonsyndromic
-like domain, four von Willebrand factor-like type hearing loss (ARNSHL) [10,15-16]. Unlike auto-
D (vWFD) domains in the zonadhesin (ZA) somal recessive mutations, which show similar
domain, and the zona pellucida (ZP) domain [2-5]. phenotypes, autosomal dominant mutations in the
Alpha-tectorin is encoded by the TECTA gene, TECTA gene result in different hearing loss
which contains 23 exons and is situated on human phenotypes depending on the protein domain that
chromosome 11q22-24 [3,6-8]. Several studies is affected [5,7,11,14,19-21].
have shown that mutations in the TECTA gene
*These authors contributed equally to this work.
Address correspondence to Un-Kyung Kim, Ph.D., Department of Biology, College of Natural Sciences, Kyungpook National
University, Daegu, 702-701, South Korea; tel 82 53 950 5353; fax 82 53 953 3066; e-mail kimuk@knu.ac.kr; or Sang-Heun Lee,
MD, Ph.D., Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Kyungpook National
University, Daegu, 702-721, South Korea; tel 82 53 420 5784; fax 82 53 423 4524; e-mail leeshu@knu.ac.kr.
0091-7370/10/0400-0380. $2.10. © 2010 by the Association of Clinical Scientists, Inc.
TECTA gene mutations in familial hearing loss 381
In this study, we identified two novel missense Results
mutations in the TECTA gene in Korean families
with ADNSHL, and we confirmed the association Mutation analysis of the TECTA gene. We
between the mutation positions in the protein identified two novel missense mutations of the
domain and their respective phenotypes. TECTA gene from the DNA samples of the 62
patients with hereditary HL who participated in
Materials and Methods this study. First, the single nucleotide change of a
cytosine for a thymine at nucleotide position 5597
Subjects. A total of 62 subjects with hereditary hearing loss
(c.5597 C>T) in exon 18 was detected in family
were recruited using the pedigree information of each
individual from the Department of Otorhinolaryngology- KNUF08. This substitution converts threonine to
Head and Neck Surgery, Kyungpook National University methionine at amino acid position 1866 (p.
Hospital. Physical examinations, including an otolaryngologic T1866M). The affected members in this family
examination, were carried out after their previous medical (II-2, III-1, III-2, and III-3) were heterozygous for
histories were reported by the subjects. According to the
inheritance patterns of hearing loss in their pedigrees, the 62
this mutation, but it was not observed in the father
individuals were classified into two groups: 43 individuals (II-1), which is consistent with the affected status
with ADNSHL and 19 individuals with ARNSHL. One in this family (Fig. 1A and 1C). This mutation was
hundred-twenty unrelated Koreans with normal hearing were not detected in 120 unrelated control subjects and
used as controls. Written informed consent was obtained
was located in the ZP domain, which is highly
from all individuals, and the study was approved by the
Institutional Review Board of Kyungpook National conserved across species (Fig. 1D).
University Hospital. Another missense mutation of TECTA was a
thymine to adenine transversion at nucleotide
Audiometric evaluation. Pure tone audiometry (PTA) was position 950 (c. 950 T>A) in exon 6, which results
performed in a sound-controlled room at frequencies ranging
from 500 to 8000 Hz, according to standard protocols. The
in a valine to a glutamic acid substitution at amino
level of hearing loss was described as follows based on the acid position 317 (p.V317E) (Fig. 2A). The patient
mean PTA calculated using the thresholds measured at 500, was heterozygous for this mutation and other
1000, 2000, and 3000 Hz: normal hearing, below 20 dB; family members were not available for study.
mild hearing impairment, 21 - 40 dB; moderate hearing
However, the inheritance pattern of hearing loss in
impairment, 41 - 70 dB; severe hearing impairment, 71 - 95
dB; and profound hearing impairment > 95 dB [22]. The this family was autosomal dominant (data not
range of hearing loss was described as follows depending on shown) and this variant is located in the highly
the PTA: low frequency, 125 - 1000 Hz; mid-frequency, 1000 conserved vWFD0 repeat of the ZA domain (Fig.
- 4000 Hz; and high frequency, 4000 - 8000 Hz. 2C). No variation was identified in any other exons
Mutation analysis. Genomic DNAs of the 62 subjects and 120
of TECTA in the patients with hereditary HL nor
controls were extracted from blood samples using a FlexiGene in 120 unrelated control subjects. This evidence
DNA kit (Qiagen, Hilden, Germany). All 23 exons and intron– supports the inference that p.V317E is a pathogenic
exon boundaries of the TECTA gene (GenBank ID mutation rather than a rare polymorphism.
NM_005422.2) were amplified by polymerase chain reaction
(PCR) in the 62 subjects. The quality of the PCR products was
monitored by electrophoresis on 2% agarose gels. Shrimp Audiological features. Fig. 1 shows the pedigree
alkaline phosphatase (USB, Cleveland, OH) and exonuclease I and the plotted PTAs of affected patients in the
(USB) were used to purify PCR products. Sequences of the KNUF08 family in which the p.T1866M mutation
purified PCR products were obtained from direct sequencing was found. None of the affected subjects had a past
using the BigDye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA). Extended products
history nor evidence of any other cause of hearing
were purified by ethanol precipitation. The 3130xl Genetic loss. The affected subjects in family KNUF08 had
Analyzer (Applied Biosystems) was used to resolve the products suffered from nonprogressive bilateral hearing loss
and the data were analyzed using Sequencing Analysis v5.2 since childhood according to their past medical
(Applied Biosystems) and Chromas Lite v2.01 (Technelysium,
histories. They all had mild to moderate sensori-
Tewantin, QL, Australia) software. Potential pathogenicity of
novel missense mutations was evaluated by SIFT (http:// neural hearing loss. The propositus, a 64-yr-old
blocks.fhcrc.org/sift/sift.html), PolyPhen (http://genetics.bwh. male with the p.V317E mutation, first visited the
harvard.edu/pph/), and multiple sequence alignment using hospital due to bilateral hearing loss at the age of 58
Sequence Viewer v6.0.1 (CLC Bio, Aarhus, Denmark).382
Annals of Clinical & Laboratory Science, vol. 40, no. 4, 2010
Fig. 1. The p.T1866M mutation detected in family KNUF08. (A) Pedigree of family KNUF08 with the p.T1866M mutation. Filled symbols indicate affected individuals. (B)
Pure tone audiograms for left and right ears of 4 affected individuals (II-2, III-1, III-2, and III-3). Numbers in parenthesis refer to age. (C) Partial DNA sequences of the TECTA
gene showing the c.5597C>T change in family member (III-1) and in a normal control. (D) Multiple alignments of the alpha-tectorin and homologous sequences in the zona
pellucida (ZP) domain partial region. The amino acid sequence of human alpha-tectorin is aligned with sequences of other species. The =arrow marks the p.T1866M mutation.
The hTECTA = alpha-tectorin of Homo sapiens; mTECTA, alpha-tectorin of Mus musculus; rTECTA, alpha-tectorin of Rattus norvegicus, and gTECTA, alpha-tectorin of Gallus
gallus.Fig. 2. (A) Partial DNA sequences of the TECTA gene showing the c.950T>A change in an affected patient vs a normal individual. The affected individual reveals heterozygosity
for valine and glutamic acid at amino acid position 317, and the normal individual is homozygous for valine. (B) Pure tone audiograms of affected individual. The dashed lines and
the solid lines show thresholds at the age of 58 and 63 yr, respectively. (C) Multiple alignments of the alpha-tectorin and homologous sequences in the von Willebrand factor-like
type D0 (vWFD0) repeat partial region. The arrow marks the position of the p.V317E mutation. The vWFD0 domain shows high conservation in the different species (see Fig. 1).
TECTA gene mutations in familial hearing loss
383384 Annals of Clinical & Laboratory Science, vol. 40, no. 4, 2010
yr. He showed bilateral mild hearing loss with and ZA domains are related to mid- and high-
down-sloping audiogram. The hearing level showed frequency hearing loss, respectively (Table 1). The
progressive deterioration at some frequencies at the p.V317E in the ZA domain was linked to progressive
age of 63 yr (Fig. 2B). high frequency hearing loss in the patient’s
audiogram and the p.T1866M in the ZP domain
Discussion showed mild-moderate hearing loss at mid-high
frequency in the affected members of the family.
In the present study, the TECTA gene was screened These results confirm earlier reports on TECTA-
in 62 Korean patients with hereditary hearing loss. related ADNSHL. Therefore, it appears that the
Two novel variants were found, p.V317E and two variations, p.V317E and p.T1866M, identified
p.T1866M, which were implicated to be patho- in this study are pathogenic missense mutations
genic missense mutations causing hearing loss. causing hearing loss in two Korean families with
Most of the missense mutations that have been ADNSHL. Further studies on the mechanical
identified in alpha-tectorin are located in the ZP association between mutation types and inheritance
domain and a few are located in the ZA domain patterns of hearing loss, and between mutation-
(Table 1). The novel variation p.V317E in the ZA affected domains and onset frequency are necessary
domain was substitution of valine with an aliphatic to elucidate the genotype-phenotype correlations
hydrophobic side chain to glutamic acid with an in patients with TECTA-related HL.
acidic side chain. Further, p.T1866M in the ZP
domain shows the substitution of the properties of Acknowledgements
amino acids, threonine to methionine, which are a
hydrophilic to a hydrophobic side chain. Thus, it This research was supported by the Basic Science
seems likely that the structural variations in the ZP Research Program through the National Research
and ZA domains might cause functional problems Foundation of Korea (NRF) funded by the
in alpha-tectorin. Ministry of Education, Science and Technology
All mutations described to date in TECTA- (2010-0028172) and by the Ministry of Education,
related ADNSHL have shown significant genotype- Science & Technology (MEST) / National
phenotype correlation between the mutated Research Foundation of Korea (NRF) through the
domains and the respective hearing loss [5-6,9,19]. Vestibulocochlear Research Center (VCRC) at
According to previous studies, mutations in the ZP Wonkwang University (R13-2002-055-00000-0).
Table 1. Missense mutations in the TECTA gene and the associated phenotypes.
Family Amino acid Exon Nucleotide Domain Frequency Phenotype Reference
origin change change
Korean V317E† 6 c.950T>A ZA a (D0) High Mild This study
Swedish C1057S 10 c.3169T>A ZA (D2) High Variable [5]
Turkish C1509G 13 c.4525T>G ZA (D4) High Mild to moderate [19]
French C1619S 14 c.4856G>C ZA (D4) High Mild to moderate [11]
Belgian L1820F 17 c.5458C>T ZPb Mid Mild to severe [7]
Belgian G1824D 17 c.5471G>A ZP Mid Mild to severe [7]
Spanish C1837G 17 c.5509T>G ZP Mid Mild to severe [21]
American C1837R 17 c.5509T>C ZP Mid-High Mild to moderate [15]
Korean T1866M† 18 c.5597C>T ZP Mid-High Mild to moderate This study
Austrian Y1870C 18 c.5609A>G ZP Mid Moderate to severe [7]
Dutch R1890C 18 c.5668C>T ZP Mid Mild to moderate [23]
Japanese R2021H 20 c.6062G>A ZP Mid Mild to moderate [14]
a ZA, zonadhesin domain; bZP, zona pellucida domain; D0-D4, von Willebrand factor-like type D0-D4 repeat.
†Novel mutations identified in this study.TECTA gene mutations in familial hearing loss 385
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