Hearing loss associated with enlarged vestibular aqueduct and Mondini dysplasia is caused by splice-site mutation in the PDS gene

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.

� 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

0378-5955/$ - see front matter � 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).

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).

J.-J. Yang et al. / Hearing Research 199 (2005) 22–30 23

Mutations of the PDS gene (MIM 605646) on chro-

mosome 7q21-34 can cause sensorineural hearing loss

with thyromegaly (Pendred syndrome, MIM 274600)

or non-syndromic hearing loss without goiter (DFNB4,

MIM 600791) (Everett et al., 1997; Li et al., 1998;

Campbell et al., 2001). PDS gene has 21 exons and con-tains an open reading frame of 2343 base pairs. The

PDS gene product, pendrin, is a highly hydrophobic

protein with 780 amino acid residues (molecular weight

86 kDa) and 11 putative transmembrane domains.

Expression of pendrin has been found in the non-sen-

sory epithelia of inner ears, thyroid folliculocytes, renal

cortical collecting ducts, placental trophoblasts and

uterine endometria (Everett et al., 1999; Royaux et al.,2000; Soleimani et al., 2001; Bidart et al., 2000; Suzuki

et al., 2002).

The hearing loss associated with PDS mutations

is thought to be related to temporal bone abnormal-

ities, ranging from isolated enlargement of vestibular

aqueduct (EVA) to Mondini dysplasia, a complex

malformation in which the normal cochlear spiral

runs of 2 1/2 turns is replaced by a hypoplastic coilof 1 1/2 turns. Both EVA and Mondini dysplasia

abnormalities can be diagnosed by either computed

tomography or magnetic resonance imaging (Phelps

et al., 1998).

In the Xenopus oocytes and Sf9 cells, pendrin has

been demonstrated to transport chloride and iodine,

and mediate the exchange of chloride and formate (Scott

et al., 1999). PDS mutations have been shown in vitro tocause the disruption of transmembrane anion/base ex-

change activity of pendrin (Scott et al., 2000). To date,

more than 66 different PDS gene mutations have been

described, and they have been found scattered all along

the 21 exons, including the flanking intronic sequences

and coding region (Tsukamoto et al., 2003).

Mutations in PDS gene have also been reported to be

associated with a wide range of phenotypes that are dif-ferent from the typical Pendred syndrome and non-syn-

dromic hearing loss with EVA (Tsukamoto et al., 2003;

Coyle et al., 1998; Van Hauwe et al., 1998; Lopez-Bigas

et al., 1999; Fugazzola et al., 2002). Therefore, identifi-

cation of PDS mutations can help elucidate the relation-

ship between mutant genotypes and phenotypes. In the

present study, we focused on whether the PDS muta-

tions were present in patients with EVA who have hear-ing loss and Mondini dysplasia. To determine the

correlation between phenotypic and genotypic data, we

have examined thePDS gene mutations in 10 prelingual

deaf patients with temporal bone abnormalities of both

EVA and Mondini dysplasia. Our data showed that all

of the patients we examined carried certain mutations

in the introns of PDS gene. Among these mutations,

six were found to be homozygous for a splicing acceptorsite in PDS gene that occurred at position

IVS7 � 2A ! G, one was found to be heterozygous

for IVS7 � 2 A ! G, two were found to be heterozy-

gous for IVS15 + 5G ! A and one heterozygous for

IVS16 � 6G! A. Our results provide evidence that

hearing loss associated with EVA and Mondini dyspla-

sia may be caused by the splice-site mutation in the

PDS gene.

2. Materials and methods

2.1. Subject selection

Ten prelingual deaf patients (from seven unrelated

families) aged between 4 and 13-years-old underwentPDS gene analysis. All 10 were found by computed

tomography of the temporal bone to have bilateral

EVA and bilateral Mondini dysplasia without goiter.

EVA was defined by an enlargement of the vestibular

aqueduct set at >1.5 mm midway between the endo-

lymphatic sac and the vestibule. Mondini dysplasia

was defined by a complex malformation in which the

normal cochlear spiral of 21/2 turns was replaced bya hypoplastic coil of 11/2 turns. Family studies were

performed on nine probands (four from unrelated fam-

ilies) with PDS mutations. Fifty randomly selected nor-

mal hearing individuals were included as control

subjects.

2.2. Detection of PDS mutation

Genomic DNA sample of each subject was ex-

tracted from whole blood using a QIAamp DNA

Blood Kit. (Qiagen) The quality and quantity of puri-

fied genomic DNA were determined by agarose gel

electrophoresis and spectrophotometry. Mutation

screenings were performed by single-strand conforma-

tion polymorphism (SSCP) and direct sequencing on

the PDS coding regions. PDS exons in DNA samplewere first amplified by polymerase chain reaction

(PCR) with intragenic primers as previously described

(Everett et al., 1997). In brief, PCR was conducted in

a volume of 25 ll which contained 100 ng of genomic

DNA, 200 lM dNTP, 0.25 units of proTaq DNA

polymerase (Promega), 200 lM intragenic primers

and 4% DMSO. The PCR products were purified

using a PCR Purification Kit (Qiagen). Reaction prod-ucts were resolved on a 8% non-denatured polyacryla-

mide gel containing 5%w/v glycerol and then

visualized by silver staining. Sequencing was per-

formed on an Applied Biosystems model 310 auto-

mated sequencer (Perkin–Elmer Corporation, Foster

City, CA). Sequence data were compared with the

published sequence of PDS gene using Sequencer 3.1

software program package (Perkin–Elmer).

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

Fig. 1. Computer tomograms of inner ear in a normal subject and 10 patients with EVA and Mondini dysplasis. Normal cochlea boxed in (a) and

vestibular aqueduct boxed in (b) in the normal subject. Mondini dysplasis boxed in (c) and enlarged vestibular aqueduct boxed in (d) in a patient.

Fig. 2. Audiograms of all patients with EVA and Mondini dysplasis shows high frequency-dominant sensorineural hearing loss: L, left ear (a);

R, right ear (b).

J.-J. Yang et al. / Hearing Research 199 (2005) 22–30 25

novo recurrence of IVS7 � 2A! G mutation. Our re-

sults found that the families carrying IVS7 � 2A ! G

mutation to have a significant haplotype between

markers D7S2420 and D7S2456 (Fig. 5). Most likely,all patients in these families had inherited the IVS7 �2A ! G mutation from a common ancestor.

4. Discussion

In 1896, Vaughan Pendred first described the Pendred

syndrome (MIM 274600) as a combination of congenitalhearing loss and goiter. PDS gene mutation is known to

be a common cause of congenital syndromic deafness,

Table 1

Mutation in the 10 prelingual deaf patients with bilateral EVA and bilateral Mondini dysplasis

Number Genotype PTA (dB) Rt. PTA (dB) Lt.

PDS 001 IVS7 � 2A!G/IVS7 � 2A!G 77 95

PDS 002 IVS7 � 2A!G/IVS7 � 2A!G 80 80

PDS 003 IVS7 � 2A!G/IVS7 � 2A!G 86 90

PDS 004 IVS7 � 2A!G/IVS7 � 2A!G 105 100

PDS 005 IVS16 � 6G! A/wt 107 83

PDS 006 IVS7 � 2A!G/IVS7 � 2A!G 80 90

PDS 007 IVS15 + 5G ! A/wt 90 83

PDS 008 IVS15 + 5G ! A/wt 62 72

PDS 009 IVS7 � 2A!G/IVS7 � 2A!G 88 85

PDS 010 IVS7 � 2A!G/wt 72 78

PTA, pure tone audiometry; Rt., right ear ; Lt., left ear.

Fig. 3. Sequence analysis of PDS genomic DNA mutation in 10 prelingual deaf patients associated with bilateral EVA and bilateral Mondini

dysplasis. Genomic sequence of the PDS gene from normal individuals (a), (d), and (f). A splice acceptor site mutation occurrs at position

IVS7 � 2A! G, including homozygous of IVS7 � 2A!G (b) and heterozygous of IVS7 � 2A! G. (c). Two of PDSmutations, IVS16 � 6G! A (e)

and IVS15 + 5G ! A (g), respectively are found to be heterozygous. Vertical arrows indicate changes of the nucleotide changed. wt: wild type.

26 J.-J. Yang et al. / Hearing Research 199 (2005) 22–30

Fig. 4. Sequence analysis of normal and IVS7 � 2A!G mutated PDS gene from RT–PCR. (a) The cDNA sequence of the region corresponds to

exons 7–9. Black bar indicates sequence of exon 8. (b) The IVS7 � 2A! G mutation in the cDNA result in deletion of exon 8. (c) Predicted protein

sequences coded from PDS gene of either normal or IVS7 � 2A !G mutation.

J.-J. Yang et al. / Hearing Research 199 (2005) 22–30 27

which accounts for 4–10% of congenital deafness in chil-

dren (Pendred, 1986; Gorlin, 1995). Park et al. (2003)

have shown that PDS gene mutations make up approx-

imately 5% of all the cases of prelingual deafness in east

Asia and 5% of recessive hearing loss in south Asia.

PDS gene product, Pendrin, is expressed in the endo-lymphatic duct and sac in the inner ear and regulates

endolymph secretion and resorption (Everett et al.,

1999). Therefore, Pendrin defects result in neuroepithel-

ial damage which in turn cause inner ear malformations

where the upper coils of the cochlea form a common

cavity (Mondini malformation) and the vestibular aque-

ducts are dilated (Johnsen et al., 1986; Phelps et al.,

1998). These inner ear anomalies lead to a generally pro-found sensorineural hearing loss, beginning prelingually

and having a mean threshold of more than 60 dB in

PTA as a mean threshold (Kabakkaya et al., 1993;

Fugazzola et al., 2002).

One previous genetic analysis has confirmed that

mutations in PDS cause a broader phenotypic spec-

trum, ranging from typical Pendred syndrome to

non-syndromic hearing loss associated with EVA(Usami et al., 1999). Campbell et al. (2001) have indi-

cated that mutations in PDS cause both Pendred syn-

drome and DFNB4. Pendred syndrome and non-

syndromic hearing loss can be differentiated clinically

by the association of goiter with the former, while

morphological abnormalities of inner ear, such as

EVA and Mondini dysplasia, are common to bothdiseases. Classically, hearing loss is congenital, and

thyromegaly is developed in the second decade (Phelps

et al., 1998; Campbell et al., 2001). Each of the 10 pa-

tients we studied exhibited severe prelingual hearing

loss with bilateral EVA and bilateral Mondini dyspla-

sia, but none of them had a goiter. Being between

4- and 13-years-old, they may have been at such an

earlier stage of disease that a had not yet developed.Another possibility is that the full expression of Pen-

dred syndrome requires gene modifications, environ-

mental factors, or a combination of these two

mechanisms. We will continue to follow these patients

into the second decade and monitor their phenotypical

changes. Whether these mutations, IVS7 � 2A ! G,

IVS16 � 6G! A and IVS15 + 5G ! A, predispose

people to the development of thyroid nodules remainsto be established.

Fig. 5. Haplotypes analysis of prelingual deaf patients with

IVS7 � 2AG mutation of PDS gene in four unrelated families. The

haplotypes linked to splice site mutation, IVS7 � 2A! G, are boxed.

x indicates crossover.

28 J.-J. Yang et al. / Hearing Research 199 (2005) 22–30

More than 66 different PDS gene mutations have

been described (Coyle et al., 1996; Everett et al., 1997;

Coucke et al., 1999; Yong et al., 2001; Tsukamoto

et al., 2003). Three frequent mutations, L236P, T416P,

and IVS8 + 1G ! A, are the most commonly reported

mutations in patients in the West, and they accountfor more than 50% of cases of Pendred syndrome (Van

Hauwe et al., 1998). Eleven mutations have been de-

tected in families with non-syndromic hearing loss asso-

ciated with EVA (Tsukamoto et al., 2003). Another

study from Japan has reported another mutation,

T410M, in non-syndromic hearing loss with EVA

(Kitamura et al., 2000). Park et al. (2003) found

H273R mutation to be a prevalent allele that could befound in a majority of PDS mutations in Korean and

Japanese populations. Nevertheless, we did not observe

any of the above mutations except for IVS7 � 2A! G.

IVS7 � 2A! G homozygous mutation was found in

6 out of the 10 patients examined in our study. This

mutation is also commonly found among different Asian

populations, such as Korean, Japanese and Chinese, but

it has not been observed in the Western populations

(Park et al., 2003). Our family studies found all ofIVS7 � 2A! G homozygous mutations to be inherited

from the same mutant allele of their IVS7 � 2A ! G

heterozygous parents, who had normal hearing. In our

family studies, we did not analyze all of the probands

because we had not collected DNA samples from

our patients. Surprisingly, we also detected the

IVS7 � 2A! G heterozygous mutation in one patient

(PDS 010) with hearing loss. Other mutations,IVS15 + 5G ! A and IVS16 � 6G ! A, represented

heterozygous mutations in which a second mutation

could not be identified in PDS gene. The significance

of IVS15 + 5GA and IVS16 � 6G ! A heterozygous

mutations is not clear but they do not seem to be com-

mon polymorphisms. Therefore, patients carrying the

heterozygous mutation are more likely to develop hear-

ing loss in the presence of additional genes or environ-mental factors.

The IVS7 � 2A ! G mutation, which changes the

conserved nucleotide of the acceptor splice site, most

probably affects the splicing of PDS gene (Krawczak

et al., 1992). Using RT–PCR, we proved that this

mutation caused the deletion of exon 8 entirely and

generated a truncated protein consisting of only 310

amino acids, a result similar to that reported forIVS8 � 4GA mutation of PDS gene, the skipping of

exon 9 of PDS gene (Massa et al., 2003). The patho-

genic potential of mutations on both IVS16 � 6G ! A

and IVS15 + 5G ! A is unknown since their effect on

splicing has not been determined. However, other

genes whose mutations occur at similar positions in

intronic regions have been found to cause various lev-

els of normal and aberrant transcripts, with the pres-ence of transcripts differing from one tissue to another

(Krawczak et al., 1992; Larriba et al., 1998). There-

fore, we presume these mutations may affect the

mRNA stability of PDS or normal protein function

of pendrin. Moreover, the possibility that the interac-

tion of PDS gene mutations with other genes resulted

in hearing loss cannot be ruled out. It is also possible

that these two nucleotide changes represent a rare oruncommon polymorphism not associated with the dis-

ease phenotype in these patients.

In haplotype analysis, we found that the patients car-

rying IVS7 � 2A ! G mutation shared the same haplo-

type. We suggest that IVS7 � 2A! G mutation may

have arisen from ancestral founder chromosomes. How-

ever, such haplotype has not been found to have any

link to IVS7 � 2A! G in different Asian populations(Korean, Chinese and Japanese) (Park et al., 2003).

We reason that IVS7 � 2A! G may be an older

J.-J. Yang et al. / Hearing Research 199 (2005) 22–30 29

founder mutation which has undergone ancestral recom-

bination events with the flanking marker.

Our evaluation of PDS mutations provide an impor-

tant base for improving the clinical diagnosis of deaf

patients with both EVA and Mondini dysplasia. More-

over, the fact that PDS gene is also expressed in thesyncytiotrophoblast cells of the placenta (Bidart et al.,

2000) shows that it may used in prenatal genetic analysis

in the future.

Acknowledgements

We thank all the subjects who participated in the pre-sent project. We also thank Chieh-Tien Shih for his

technical assistance. This work is supported by National

Science Council, ROC (NSC 91-2745-P-040-002, and

NSC 91-2320-B-040-015).

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