International Journal of Pediatric Otorhinolaryngology 77 (2013) 1495–1499

A Novel mutation in the SLC26A4 gene in a Chinese family withPendred syndrome

Chun-Jui Huang a,b, Tsun-Hsing Lei a, Wei-Lun Chang a, Tzong-Yang Tu c,An-Suey Shiao b,c, Chih-Yang Chiu d, Tjin-Shing Jap a,b,*a Division of Endocrinology & Metabolism, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwanb National Yang-Ming University School of Medicine, Taipei, Taiwanc Department of Otorhinolaryngology – Head and Neck Surgery, Taipei Veterans General Hospital, Taipei, Taiwand Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan

A R T I C L E I N F O

Article history:

Received 16 April 2013

Received in revised form 15 June 2013

Accepted 18 June 2013

Available online 6 July 2013

Keywords:

Pendred syndrome

SLC26A4

Pendrin

Enlarged vestibular aqueduct

Deafness

A B S T R A C T

Objective: To investigate the mutations in the SLC26A4 gene in a Chinese patient with Pendred

syndrome.

Methods: The diagnosis of Pendred syndrome was confirmed by the family history, pure tone audiogram,

perchlorate discharge test (PDT), and computed tomography (CT) of the temporal bone. DNA extraction,

PCR and DNA sequencing were performed according to standard procedures. Mutations in the SLC26A4

gene were compared with 100 unrelated subjects to exclude common polymorphism. Splice-site

mutation was further confirmed by restriction enzyme length polymorphism (RFLP) with the specifically

designed primers.

Results: The proband presented with typical features of bilateral sensorineural deafness since childhood

and goiter development in the early adulthood. Thyroid studies disclosed euthyroidism with elevated

thyroglobulin, but negative for PDT. Marked enlargement of bilateral vestibular aqueduct (>1.5 mm)

was found by CT of the temporal bone. A novel SLC26A4 splice-site mutation c.1263+1G>A

(IVS10+1G>A) was identified in compound heterozygosity with the missense mutation c.1079C>T

(p.A360V) in the proband. Both mutations were not found in the 100 unrelated Chinese.

Conclusions: Our results support previous findings that Pendred syndrome can be caused by compound

heterozygous mutation in the SLC26A4 gene, in which IVS10+1G>A is a novel pathogenic mutation.

� 2013 Elsevier Ireland Ltd. All rights reserved.

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International Journal of Pediatric Otorhinolaryngology

jo ur n al ho m ep ag e: ww w.els evier . c om / lo cat e/ i jp o r l

1. Introduction

Pendred syndrome (PS) is an autosomal recessive disordercharacterized by congenital sensorinueral deafness, goiter, andpartial iodine organification defect [1]. The hearing loss is oftenprelingual, profound and with a fluctuating course [1]. Themanifestations of thyroid usually develop in the childhood oradulthood later after hearing loss [2]. The spectrum of thyroiddysfunction ranges from euthyroid to hypothyroidism, dependingon the degree of iodine intake [2–4]. A positive perchloratedischarge test can confirm the defect in iodine organification [4].Specific inner ear malformation detected by computed tomogra-phy (CT) of the temporal bones is essential for the diagnosis of PS[5,6].

* Corresponding author at: Division of Endocrinology & Metabolism, Department

of Medicine, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei,

112, Taiwan. Tel.: +886 2 2875 7516; fax: +886 2 2874 5674.

E-mail address: tsjap@vghtpe.gov.tw (T.-S. Jap).

0165-5876/$ – see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ijporl.2013.06.017

It was not until a century after the first description of PS that thedisease-causative gene, Pendred syndrome (PDS) gene, laterrenamed as SLC26A4, was found and localized to the chromosome7q [7–9]. This gene encodes the protein, pendrin, which is amultifunctional anion transporter expressed in the thyroid, kidney,and inner ear. Mutation in the PDS gene may result in defectivefunctions in the anion transport in the thyroid and inner ear,causing deafness or goiter [10].

According to previous literature, high heterogeneity andethical differences were noted in the PDS mutations. Althoughmissense mutations account for the majority of mutationsworldwide, a splice-site mutation, c. 919-2A>G (IVS7-2A>G)was reported to account for more than 80% of mutations inTaiwan [11,12]. So far, more than 240 mutations have beenidentified in the SLC26A4 gene of patients with PS (http://www.healthcare.uiowa.edu/labs/pendredandbor/slcMuta-tions.htm). Genetic analysis in our patient with PS may expandthe mutation spectrum of SLC26A4 gene in the Chinesepopulation, thus providing important information on clinicaldiagnosis and genetic counseling.

Fig. 1. Pure tone audiogram of the proband revealed near total sensorineural

hearing loss of left ear and 85 dB sensorineural hearing loss of right ear.

Table 1Thyroid autoantibodies of the proband and her parents.

Proband Mother Father

Anti-TG Ab (U/ml) 41.4 36.0 37.5

Anti-TPO Ab (U/ml) 42.7 28.9 14.6

TSH R Ab (%) <3 <3 13

Thyroglobulin (Tg) >300 30.7 12.6

Anti-TG Ab, Anti-thyroglobulin antibody (normal < 60 U/ml); Anti-TPO Ab, anti-

thyroid peroxidase antibody (normal < 60 U/ml); TSH R Ab, thyroid-stimulating

hormone receptor antibody (normal < 10%).

Fig. 2. CT of temporal bone disclosed marked enlargement of bilateral vestibular

aqueduct (Right: 0.59 cm, left: 0.58 cm, normal � 0.15 cm) [15]. Arrow: vestibular

aqueduct.

C.-J. Huang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1495–14991496

2. Patients and methods

2.1. Subject and clinical evaluation

The proband is a case in Taipei Veterans General Hospital.Demographic data and a detailed history of her family wererecorded. Hearing impairment was diagnosed by otorhinolaryn-gologist with pure tone audiometry. Inner ear malformation wasdetected by CT of temporal bone. Thyroid studies including thyroidfunction, thyroglobulin, thyroid autoantibodies, and thyroidultrasonography were performed. An oral perchlorate dischargetest (PDT) was completed with the following protocol. 0.925 mBqradioactive iodine (iodine 131) was first administered for scanningof basal radioiodine uptake. Oral 1.0 g KCIO4 was then given 2 hlater with a follow up scan. A decrease of more than 10% ofradioactivity at 2 h was considered positive. This study wasapproved by the institutional review board. Informed consent wasobtained from all individuals.

2.2. Definition of Pendred syndrome

The diagnosis of PS was based on the classical findings ofsensorineural deafness, enlargement of vestibular aqueduct (EVA)and goiter of non-endemic and non-autoimmune in origin.

2.3. Genetic analysis of the SLC26A4 gene

The genomic DNA was isolated from the EDTA-contained wholeblood with the GFX Genomic Blood DNA Purification Kit (AmershamBiosciences, Piscataway, NJ, USA). For the amplification of the 20coding exons (numbered 2�21) of the SLC26A4 gene, polymerasechain reaction (PCR) of peripheral leukocytes was performed withprimers and conditions as previously described [13]. The PCRreactions were completed in a 25 ml reaction mixture containing200 ng of genomic DNA, 2.0 mmol/L MgCl2, 0.2 mmol/L of eachdeoxynucleoside triphosphate (dNTP), 0.15 mol/L of each primer, 1�reaction buffer, and 1 unit of FastStart Taq DNA polymerase (Roche,Indianapolis, IN). PCR products were purified by spin column usingthe GFX PCR DNA and Gel Band Purification Kit (AmershamBiosciences, Piscataway, NJ). DNA was sequenced by automatedDNA sequencing analysis with fluorescence-labeled dideoxytermi-nators (BigDye Terminator V3.1 Cycle Sequencing Kits, AppliedBiosystems, Foster City, CA) according to the manufacturer’srecommendations (ABI 377-36 autosequencer, Applied Biosystems)

2.4. Mutation confirmation

One hundred unrelated normal Chinese subjects recruited in aprevious study served as control to determine whether anysequence variation might be a common polymorphism [14]. Thepresence of the novel mutation in our proband and the absence ofthis mutation in 100 controls were confirmed by restrictionenzyme length polymorphism (RFLP) with the modified primers.The modified primer introduces a base substitution at nucleotideposition 1262, thus creating an artificial cutting site to allow onlythe wild-type sequence to be digested by MspI. The digestedproducts were then analyzed by electrophoresis through a 12.5%polyacrylamide gel with fragments visualized by silver staining kit(Amersham Pharmacia Biotech AB, Uppsala, Sweden).

3. Results

3.1. Clinical, audiological and imaging data

A 21 year-old female patient was admitted due to sudden onsetof hearing deterioration of left ear. She had suffered from bilateral

hearing impairment since childhood. The hearing condition wasnormal in her parents. Eardrums were intact upon otoscopic exam,but pure tone audiometry disclosed near total sensorineuralhearing loss of left ear and 85 dB sensorineural hearing loss of rightear (Fig. 1). Thyroid gland was enlarged (Grade II), but thyroidfunction was normal (free T4 0.97 ng/dl, normal: 0.8–1.9; TSH1.290 uIU/ml, normal: 0.4–4.0) and thyroid autoantibodies wereabsent, except for elevated serum thyroglobulin (Tg) level(Table 1). Marked enlargement of bilateral vestibular aqueduct,compatible with the typical finding of large vestibular aqueductsyndrome was detected by CT of temporal bone (Fig. 2) [15]. Theresult of PDT was negative, with 9% baseline I131 uptake, 8.3%uptake at 1 h, and 8.6% at 2 h. Her hearing impairment got partially

Fig. 3. SLC26A4 mutations in the family with PS.

C.-J. Huang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1495–1499 1497

improved after treatment with intravenous dexamethasone (5 mgbid � 3 days, followed by 5 mg qd � 2 days) and plasma expander.After the episode, sudden worsening of sensorineural hearing losshappened occasionally and became refractory to the therapy ofintravenous steroid, plasma expander, and intra-tympanic steroidinjection.

3.2. Genetic analysis

To confirm the diagnosis of PS, further genetic analysis wasperformed. Compound heterozygous mutation in the SLC26A4gene was found in the proband: the missense mutation c.1079C>T(p.A360V) in exon 9, resulting in the replacement of alanine byvaline at codon 360, was from the mother; the splice-site mutationc.1263+1G>A (IVS10+1G>A) in intron 10 was from the father(Fig. 3). IVS10+1G>A was confirmed to cause incorrect splicing byrestriction fragment length polymorphism (RFLP) as shown inFig. 4.

Fig. 4. After PCR with the modified cutting-site primer that introduces a base

substitution at nucleotide position 1262, further digestion with MspI yielded two

restriction products of 171 bp and 20 bp in the wild type allele and 191 bp in the

IVS10 + 1G > A mutant allele, thus creating two visualized bands on electrophoresis

in the proband.

4. Discussion and conclusions

To our knowledge, the mutation, IVS10+1G>A, was the firstreport in patients with Pendred syndrome. According to theliterature, the type and prevalence of SLC26A4 mutations varieswidely among different ethnic groups. The mutation spectrum andnucleotide changes of the SLC26A4 gene in the Chinese populationwere summarized in Fig. 5 [11,12,14,16–35]. A total of 134 variantshave been reported with the majority located in the coding regions(n = 115) of exon 2–21. Some nucleotide changes in the intronsfound close to the coding regions were considered to be pathogenic(n = 19), including the current novel mutation, IVS10+1A>G(c.1263+1G>A). IVS7-2A>G (c.919-2A>G) followed by p.H723R(c.2168A>G) were the most frequently found mutations in theChinese population, accounting for more than 80% of the cases[11,12,14,16–22]; in the western population, the 10 most commonlydetected mutations were p.L236P, p.T416P, IVS8+1G>A, p.E384G,p.L445W, p.T410M, p.G209V, p.V138F, p.Y530H, and p.L597S,accounting for nearly 50% of mutations [36–43].

IVS10+1G>A was not identified in 100 Chinese healthyindividuals, indicating the mutation is not a common polymor-phism. There were no other mutations found in the other codingregions. IVS10+1G>A was located in the typical eukaryoticconsensus sequences of the intron; such mutation in the conservedsequence of splice acceptor site is considered to result in abnormalsplicing. Based on the above reasons, we thought that this novelmutation plays a pathogenic role in our patient with PS.

Our case presented with typical clinical features of PS includingcongenital, profound bilateral sensorineural deafness with afluctuating course and development of goiter in the earlyadulthood. The euthyroid goiter in our case is not likely to beendemic goiter because iodine intake in Taiwan is sufficient oreven higher than recommended [44]; in this condition, hypothy-roidism infrequently develops [45]. Elevated serum Tg wascommonly noted in the patients with PS [46], like our current case.

Besides from the clinical manifestations of thyroid disordersand deafness, specific inner ear malformation including enlarged

Fig. 5. Summary of mutation spectrum and nucleotide changes of the SLC26A4 gene in the Chinese population. A total of 134 variants have been reported with the majority

located in the coding regions (n = 115) of exon 2–21 and 19 variants located in the intron near the coding regions, including the current novel mutation c.1263 + 1G > A. In

ethnic Chinese, c.919-2A > G together with c.2168A > G in the area of hot spot mutation, accounted for more than 80% of SLC26A4 mutations. (n): reference number; denotes

hot spot mutation; *current novel mutation. Mutations in the introns are in italic style and bold. Mutations found only in ethnic Chinese were underlined.

C.-J. Huang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1495–14991498

vestibular aqueduct and/or Mondini cochlea is essential in thediagnosis of PS [5,6]. However, this inner ear malformation wasalso found in patients with non-syndromic enlarged vestibularaqueduct (NSEVA), which can be associated with zero, single ordouble mutations [41,47]. In contrast, PS is associated withbiallelic mutation [41,47], making genetic analysis very importantin facilitating the diagnosis and differentiating between PS, NSEVA,and simple goiter with NSEVA. A novel mutation, IVS10+1G>Acompound with p.A360V was found in our patient, furtherconfirming the diagnosis of PS.

In conclusion, IVS10+1G>A is a novel mutation in the SLC26A4gene and should be listed as one of the genetic studies inconfirming the diagnosis of PS.

Conflict of interest

None.

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