Hearing loss associated with enlarged vestibular aqueduct and Mondini dysplasia is caused by splice-site mutation in the PDS gene Jiann-Jou Yang a,b , Chin-Chu Tsai a , Hsiu-Mei Hsu a , Jiun-Yih Shiao c , Ching-Chyuan Su b,d , Shuan-Yow Li a, * a Genetics Laboratory and Department of Life Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Road, Taichung 402, Taiwan, ROC b Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC c Department of Otorhinolaryngology, Taichung VeteransGeneral Hospital, Taichung, Taiwan, ROC d Tian-Sheng Memorial Hospital, Tong Kang, Pin-Tong, Taiwan, ROC Received 19 February 2004; accepted 9 August 2004 Available online 6 October 2004 Abstract Recessive mutations of PDS gene are the common causes of Pendred syndrome and non-syndromic hearing loss associated with temporal bone abnormalities ranging from isolated enlargement of the vestibular aqueduct (EVA) to Mondini dysplasia. In this study we evaluate the relationship between EVA and Mondini dysplasia in 10 prelingual deaf patients and PDS gene mutation. One of three mutations, IVS7 2A ! G, IVS16 6G ! A or IVS15 + 5G ! A, was identified in the PDS gene in each patient. In family studies of four probands with the IVS7 2A ! G mutation, we found that this mutation was inherited from the same mutant alleles of parental origin. The effect of IVS7 2A ! G mutation on PDS gene expression was determined by reverse tran- scription and polymerase chain reaction (RT–PCR). Sequencing of the RT–PCR products revealed that the PDS transcripts from the allele with IVS7 2A ! G mutation lose the entire exon 8, resulting in a joining of exons 7 and 9. Deletion of the exon 8 results in frameshift and premature termination of translation. Haplotype analysis showed a significant haplotype shared among the family members carrying IVS7 2A ! G mutation, suggesting that they may be derived from a common ancestor. Our results provide evidence that hearing loss with EVA and Mondini dysplasia may be caused by splice-site mutation in the PDS gene. Ó 2004 Elsevier B.V. All rights reserved. Keywords: PDS; EVA; Mondini dysplasia; Prelingual deafness; Pendrin syndrome; Hearing loss 1. Introduction Hearing loss is a common sensory disorder. The inci- dence of congenital hearing loss is estimated at 1 in 1000, with about an equal number of cases attributed to environmental and genetic factors (Morton, 1991; Gorlin, 1995). Environmental causes for hearing loss are probably in decline as better therapies for bacterial and viral infections (e.g. vaccines) are created and acoustic trauma in the workplace is recognized and pre- vented. Ototoxic drugs (e.g. aminoglycosides) are now also being avoided in genetically susceptible individuals (Morton, 2002). Of the hearing loss disorders attributable to genetic causes, nearly 70% are classified as non-syndromic and the remaining 30% syndromic. (Resendes, 2001). 0378-5955/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2004.08.007 Abbreviations: EVA, enlargement of the vestibular aqueduct; IVS, intervening sequence; RT–PCR, reverse transcription and polymerase chain reaction; DFNB, deafness, autosomal recessive; SSCP, single- strand conformation polymorphism; PCR, polymerase chain reaction; PTA, pure tone audiometry * Corresponding author. Tel.: +886 4 2473 0022x1800; fax: +886 4 2475 7412. E-mail address: [email protected](S.-Y. Li). www.elsevier.com/locate/heares Hearing Research 199 (2005) 22–30
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www.elsevier.com/locate/heares
Hearing Research 199 (2005) 22–30
Hearing loss associated with enlarged vestibular aqueduct andMondini dysplasia is caused by splice-site mutation in the PDS gene
Jiann-Jou Yang a,b, Chin-Chu Tsai a, Hsiu-Mei Hsu a, Jiun-Yih Shiao c,Ching-Chyuan Su b,d, Shuan-Yow Li a,*
a Genetics Laboratory and Department of Life Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Road,
Taichung 402, Taiwan, ROCb Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC
c Department of Otorhinolaryngology, Taichung VeteransGeneral Hospital, Taichung, Taiwan, ROCd Tian-Sheng Memorial Hospital, Tong Kang, Pin-Tong, Taiwan, ROC
Received 19 February 2004; accepted 9 August 2004
Available online 6 October 2004
Abstract
Recessive mutations of PDS gene are the common causes of Pendred syndrome and non-syndromic hearing loss associated with
temporal bone abnormalities ranging from isolated enlargement of the vestibular aqueduct (EVA) to Mondini dysplasia. In this
study we evaluate the relationship between EVA and Mondini dysplasia in 10 prelingual deaf patients and PDS gene mutation.
One of three mutations, IVS7 � 2A ! G, IVS16 � 6G ! A or IVS15 + 5G ! A, was identified in the PDS gene in each patient.
In family studies of four probands with the IVS7 � 2A! G mutation, we found that this mutation was inherited from the same
mutant alleles of parental origin. The effect of IVS7 � 2A !G mutation on PDS gene expression was determined by reverse tran-
scription and polymerase chain reaction (RT–PCR). Sequencing of the RT–PCR products revealed that the PDS transcripts from
the allele with IVS7 � 2A !G mutation lose the entire exon 8, resulting in a joining of exons 7 and 9. Deletion of the exon 8 results
in frameshift and premature termination of translation. Haplotype analysis showed a significant haplotype shared among the family
members carrying IVS7 � 2A ! G mutation, suggesting that they may be derived from a common ancestor. Our results provide
evidence that hearing loss with EVA and Mondini dysplasia may be caused by splice-site mutation in the PDS gene.
24 J.-J. Yang et al. / Hearing Research 199 (2005) 22–30
2.3. mRNA extraction and reverse transcription
Total RNA was isolated from peripheral blood
lymphocytes using the Total RNA Extraction Miniprep
System (VIOGEN) according to manufacturer�s instruc-tion. cDNA was synthesized in a reaction of 20 ll whichcontained 2–5 lg RNA, random hexamer primer, and
200 units Improm-IITM Reverse Transcriptase (Promega)
according to the manufacturer�s instructions.
2.4. cDNA analysis
A fragment containing exons 7, 8 and 9 of the PDS
gene was amplified from cDNA by PCR with primersfrom the coding region (forward 5 0-gtgaggtacttggca-
gatcctt-3 0 and reverse 5 0-cctactgacactgcaatagc-3 0). PCR
reactions were performed in a 25 ll reaction mixture
which contained 1 mM Tris–HCl (pH 9.0), 5 mM
KCl, 150 lM MgCl2, 200 lM dNTP, 0.25 units pro
Taq DNA polymerase (Promega), 100 ng of cDNA,
and 200 lM forward and reverse primers. PCR products
were purified using Gel-MTM Gel Extraction System(VIOGEN). Predenaturation at 95 �C for 5 min, cycled
35 times through the following procedure: denaturation
at 95 �C for 30 s, annealing at 58 �C for 35 s, extension
at 72 �C for 45 s and a final extension step at 72 �C for 7
min. Direct automatic sequencing of PCR products was
performed with the forward and reverse primers using
an Applied Biosystems model 310 automated sequencer.
(Perkin–Elmer) Sequence data were compared as men-tioned above.
2.5. Haplotype analysis
Five polymorphic DNA markers covering the 7q
PDS regions (D7S2549, D7S2420, D7S496, D7S2459,
and D7S2456) were used in the haplotype analysis.
Information about sequence and amplification condi-tions of these markers were obtained from the CEPH-
Genethon set or Genome Data Base (Dib et al., 1996;
http://www.gdb.org). One hundred ng of genomic
DNA samples obtained from siblings and parents of
the study subjects were analyzed with PCR amplification
using fluorescence-tagged primers for each polymorphic
marker. Genotypes were scored semiautomately with an
ABI310 sequencer using GenescanTM/GenotyperTM
(Perkin–Elmer). The haplotype of each study subject
was reconstructed using the GENEHUNTER program
(Kruglyak et al., 1996).
3. Results
Ten prelingual deaf patients from seven unrelatedfamilies were found to have bilateral EVA and bilateral
Mondini dysplasia. These patients were found by com-
puted tomography to have temporal bones abnormali-
ties (Fig. 1) and by pure tone audiometry (PTA) to
have hearing loss to frequencies between 250 and 8000
Hz with a mean threshold of more than 62 dB in both
right and left ears using (Fig. 2).The patients were screened for the mutations in PDS
gene, which cover 21 exons including the flanking intro-
nic sequences and PDS coding region by PCR amplifica-
tion and SSCP. Abnormal migration of SSCP bands
were detected in all of 10 deaf patients (data not shown).
The nature of the abnormal migration was determined
by direct DNA sequence analysis. One of the three
mutations, IVS7 � 2A ! G, IVS16 � 6G ! A andIVS15 + 5G ! A, were identified in the PDS gene in
each patient. The observed mutations are summarized
in Table 1 and Fig. 3. These three mutations were not
detected in the 50 normal hearing control individuals.
Of the 10 patients, PDS gene from 6 patients were
found to be homozygous for a splice acceptor site muta-
tion that occurred at position IVS7 � 2A ! G (Fig.
3(b)), 1 heterozygous mutation for IVS7 � 2A ! G(Fig. 3(c)), 1 heterozygous mutation for IVS16 �6G! A (Fig. 3(e)), and 2 heterozygous for IVS15 +
5G! A (Fig. 3(g)). We also performed family studies
on nine probands with IVS7 � 2A! G mutation. All
were found to be inherited from the same mutant alleles
of their heterozygous parents who had normal hearing
ability (data not shown).
To study whether the PDS mutations (IVS7 �2A! G, or IVS16 � 6G ! A, or IVS15 + 5G! A)
had any effect on the mRNA expression, the levels of
PDS transcripts were determined by a combination of
reverse transcription and PCR (RT–PCR). In
IVS7 � 2A! G mutation, the RT–PCR product gener-
ated by primers located between exons 7 and 9 showed a
212 bp fragment, which is smaller than the 295 bp PCR
fragment obtained from a normal control (data notshown). Sequencing of the 212 bp PCR products re-
vealed that the PDS transcripts from the allele with
IVS7 � 2A! G mutation skipped exon 8 entirely,
resulting in a joining of exons 7 and 9 (Fig. 4(a) and
(b)). The deletion of exon 8 generated a new stop codon
at position 311, which might result in a premature trun-
cated protein of only 310 amino acids (Fig. 4(c)). How-
ever, we did not find any abnormal RT–PCR product bysize of sequence analysis of PDS gene from patients car-
rying mutations of either IVS16 � 6G ! A or IVS15 +
5G! A (data not shown).
Disease-associated haplotypes across the PDS gene
region at chromosome 7q were determined by typing
five microsatellites, including D7S2549, D7S2420,
D7S496, D7S2459, and D7S2456. IVS7 � 2A ! G
linked haplotypes of five microsatellites collected fromsiblings and parents in four unrelated families and ana-
lyzed to distinguish between founder effects and de