Analysis of the Thyroid Phenotype in 42 Patients with Pendred Syndrome and Nonsyndromic Enlargement of the Vestibular Aqueduct

Abstract

Background:

Pendred syndrome (PS), a recessive disorder caused by mutations in the SLC26A4 (PDS) gene, is associated with deafness and goiter. SLC26A4 mutations have also been identified in patients exhibiting isolated sensorineural hearing loss without apparent thyroid abnormality (nonsyndromic enlargement of the vestibular aqueduct; nonsyndromic EVA). Our aim was to describe systematically the thyroidal phenotypes and the SLC26A4 genotypes of patients presenting with PS or nonsyndromic EVA.

Methods:

Nineteen patients with PS and 23 patients with nonsyndromic EVA, aged 5–53 years, were included. They underwent thyroid evaluation (physical examination, biological thyroid function tests, measurement of thyroglobulin level, thyroid ultrasonography, and thyroid ¹²³I scintigraphy with perchlorate discharge test), otological evaluation, and SLC26A4 mutation screening.

Results:

In 19 patients with PS, goiter was identified in 15 (79%) and hypothyroidism in 15 (79%); hypothyroidism was subclinical in four patients and congenital in six patients. The perchlorate discharge test (PDT) was positive in 10/16 (63%). Morphological evaluation of the inner ear using MRI and/or CT showed bilateral EVA in 15/15 PS patients. Mutation screening revealed two SLC26A4 mutant alleles in all 19 PS patients that were homozygous in two families and compound heterozygous in 12 families. In the 23 patients with nonsyndromic EVA, systematic thyroid evaluation found no abnormalities except for slightly increased thyroglobulin levels in two patients. SLC26A4 mutations were identified in 9/23 (39%). Mutations were biallelic in two (compound heterozygous) and monoallelic in seven patients.

Conclusion:

The thyroid phenotype is widely variable in PS. SLC26A4 mutation screening is needed in patients exhibiting PS or nonsyndromic EVA. PS is associated with biallelic SLC26A4 mutations and nonsyndromic EVA with no, monoallelic, or biallelic SLC26A4 mutations. Systematic thyroid evaluation is recommended in patients with nonsyndromic EVA associated with one or two SLC26A4 mutations. We propose using a combination of three parameters to define and diagnose PS: (i) sensorineural deafness with bilateral EVA; (ii) thyroid abnormality comprising goiter and/or hypothyroidism and/or a positive PDT; (iii) biallelic SLC26A4 mutations.

Introduction

Pendred syndrome (PS) (OMIM 274600) is an autosomal recessive disorder that is characterized by sensorineural deafness and goiter (1). Deafness is usually prelingual, severe to profound, fluctuating, and progressive, and is associated with typical inner ear abnormalities (2). Enlargement of the vestibular aqueduct (EVA) (OMIM 600791) and its contents, the endolymphatic duct and sac, is identified in 80% to 100% of patients by computed tomography (CT) or magnetic resonance imaging (MRI) (3,4). The Mondini cochlear malformation, comprising an incomplete partition of the cochlea, is present in a minority of patients (20% of patients on CT) (5). PS represents 4% to 10% of all causes of inherited deafness (1,6). The classic thyroid phenotype comprises goiter associated with a partial iodine organification defect, which can be identified by a positive perchlorate discharge test (PDT) (7,8). Some patients also exhibit mild hypothyroidism. The phenotype is highly variable, and none of those classic features seems to be constant (9,10).

PS is caused by biallelic mutations in the SLC26A4 gene (formerly called PDS) (11), which codes for pendrin, a transmembrane protein that acts as a Na⁺-independent exchanger for anions such as Cl⁻, I⁻, HCO₃ ⁻, OH⁻, and formate (12). Pendrin is expressed mainly in the inner ear, thyroid, and kidney. In the thyroid, pendrin is located in the apical membrane of the thyrocyte (13) and mediates Cl⁻ and I⁻ transport, permitting apical iodide efflux into the follicular lumen (14,15). In the inner ear, pendrin is expressed in the endolymphatic duct and sac (16). It acts as an exchanger of Cl⁻ and HCO₃ ⁻ and contributes to pH and ionic homeostasis of the endolymph. Loss of pendrin activity during inner ear development leads to a hydrops of the endolymphatic system, associated with acidification of the endolymph, loss of the endolymphatic potential, and failure to acquire normal hearing (17). In the kidney, pendrin is expressed in cells of the cortical collecting tubules, distal convoluted tubules, and connecting tubules, and it mediates Cl⁻/HCO3⁻ exchange (18,19). However, loss of pendrin does not affect kidney function or arterial pH under basal conditions, probably because of the presence of redundant mechanisms of ion exchange (20). More than 371 different SLC26A4 mutations have been reported (www.biobase-international.com/product/hgmd).

SLC26A4 mutations have also been described in patients exhibiting sensorineural hearing loss and EVA without any thyroid abnormality (nonsyndromic EVA) (21,22). Monoallelic or biallelic mutations were noted in 40% to 60% of Caucasian patients with nonsyndromic EVA, either in familial or isolated forms (23,24), and in 80% to 90% of Asian patients (25 –27). The reason why the thyroid is not affected in the nonsyndromic forms of EVA with SLC26A4 mutation(s) is not clear.

We performed a systematic thyroid evaluation of 19 patients with PS and 23 patients with nonsyndromic EVA in order to improve the definition of the thyroid phenotype and to determine whether there is a genotype/phenotype correlation.

Materials and Methods

Patient recruitment

The study was approved by the ethics committee of Lille University Hospital in France. Written informed consent was obtained from all subjects or their parents or legal guardians. We recruited two groups of patients. In the first group, we recruited 28 patients who were referred to pediatricians, otolaryngologists, or endocrinologists with suspected PS, in particular patients exhibiting profound sensorineural hearing loss associated with goiter and/or hypothyroidism. We also recruited five of their deaf siblings. A complete medical history and examination was obtained for each patient to exclude other causes of thyroid disorders or deafness. Available thyroid, otological, and morphological data were collected. Missing phenotypic evaluations were performed as described below, whenever possible (e.g., missing thyroid data were not evaluable in patients who underwent previous surgical thyroidectomy). Mutation screening of the SLC26A4 gene was performed. Since the PS thyroid phenotype is poorly defined, we excluded 14 patients who were neither carrying SLC26A4 mutant allele(s) nor exhibiting EVA from the study. We finally retained 19 patients in the first group, and their results were analyzed retrospectively.

In the second group, we recruited 23 consecutive patients who were referred to otolaryngologists with deafness associated with confirmed EVA in at least one ear. No patient was identified via newborn hearing screening. They all underwent a complete otological evaluation. Thyroid evaluation and SLC26A4 mutation screening were performed prospectively as described below. Most patients came from Lille University Hospital, except for five patients who came from other French university hospitals (Hôpital Saint Vincent de Paul in Paris, Tours, Toulouse, and Bordeaux).

Otological evaluation

Patients underwent a complete audiological evaluation including otoscopic examination, pure-tone audiometry or conditioned audiometry, and impedance audiometry with and without brainstem auditory evoked potentials. Morphological evaluation of the inner ear was performed using high-resolution temporal bone CT and/or high-resolution MRI of the inner ear. EVA was defined as a vestibular aqueduct diameter exceeding 1.5 mm at the midpoint between the posterior cranial fossa and the vestibule of the inner ear.

Thyroid evaluation

Thyroid evaluation included physical examination by an endocrinologist, biochemical thyroid function tests (thytrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), and thyroglobulin levels), detection of antibodies (antithyroglobulin, antithyroperoxidase), thyroid ultrasonography, and thyroid ¹²³I scintigraphy with the PDT. Subclinical hypothyroidism was defined as an elevated TSH level (>4 mIU/L) associated with fT3 and fT4 levels within the normal range. Thyroid volume was determined ultrasonographically by adding the volume of both lobes (width×length×thickness×0.52). Goiter was defined as a thyroidal volume >20 mL in adult men and >18 mL in adult women. For children, goiter was defined as a thyroidal volume superior to one standard deviation (SD) above the mean volume reported by Chanoine et al., who studied a pediatric population living in Brussels, a moderately iodine deficient area that is very close to our center (28). In PDT, potassium perchlorate was orally administered (1 g potassium perchlorate in adults, 15 mg/kg in children) 180 min after the radioiodine (¹²³I) and activity was measured in the thyroid at 15, 30, 45, and 60 min after perchlorate administration. A positive PDT was defined as >10% discharge of radioiodine. Levothyroxine treatment was discontinued prior to testing.

Genetic analysis

Mutation screening of the SLC26A4 gene was performed using denaturing high performance liquid chromatography (DHPLC). Mutations were identified using bidirectional direct sequencing of the 20 coding exons of the gene (exons 2–21) and its flanking regions. Written informed consent was obtained from all the patients or their parents or guardians.

DNA was extracted from whole blood samples using the Nucleon BACC 3 kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions. The 20 SLC26A4 coding exons (exons 2–21) and their flanking splicing sites were amplified by polymerase chain reaction (PCR) using previously described intronic primers (11,29 –31). Touchdown PCR (Biometra Thermocycler, Goettingen, Germany) was used for exon amplification. After the PCR, the samples were pooled with two parts of unknown sample and one part of wild-type sample, and the heteroduplexes were allowed to form by denaturing at 98°C for 5 min and cooling at a rate of 0.5°C every 20 sec to 50°C. DHPLC analysis was performed using the Wave System 35000HT (Transgenomic, Omaha, NE). The amplicons were analyzed using the Wavemaker Software (Transgenomic) to determine the optimal analysis temperature(s).

PCR products showing variant patterns were purified with the NucleoSpin Extract (Machery-Nagel, Düren, Germany) and sequenced directly on a CEQ 800 (Beckman Coulter, Brea, CA) automated sequencer using the CEQ Dye Terminator Cycle Sequencing kit (Beckman Coulter). We also directly sequenced the 20 SLC26A4 coding exons of patients showing one or no DHPLC variant pattern to control the sensitivity of our technique.

Results

Forty-two patients from 37 unrelated families were included in the study: 19 patients with PS (14 families) and 23 patients with nonsyndromic EVA (23 families).

Patients with PS (19 patients, 14 families)

The mean age was 25.2 years (range 5–42; 10 males and 9 females; Table 1). Morphological evaluation of inner ears was performed in 15 patients. All 15 exhibited bilateral EVA on the CT scan or enlargement of the endolymphatic duct/sac on the MRI. CT alone was performed in seven patients, MRI alone in two, and both CT and MRI in six; the combined CT and MRI identified bilateral EVA and enlargement of the endolymphatic duct/sac in these six patients. Bilateral cochlear dysplasia was observed in seven patients.

Table 1.

Clinical Characteristics and Genetic Analysis of Patients with Pendred Syndrome

											SLC26A4 genotype
No	Family	Sex	Age	Recruitment	Inner ear	Goiter	Hypo	Diagnosis of hypothyroidism	Tg (ng/mL)	PDT	Allele 1	Allele 2
1	1	M	41	Endo	EVA^a	Yes, nodular	Yes	<1 year	ND	Positive	p.Val138Phe	p.Leu445Trp
2	1	M	37	Endo	ND	Yes, nodular	Yes	<1 year	244	Positive	p.Val138Phe	p.Leu445Trp
3	2	F	23	Otolaryn	EVA	Yes, nodular	Yes	16 years	580	Positive	p.Val138Phe	p.Glu384Gly
4	3	F	26	Endo	ND	Yes, nodular	Yes	Congenital	ND	Positive	p.Glu384Gly	p.Leu445Trp
5	3	M	42	Endo	EVA	Yes, nodular	Yes	5 years	1212	Positive	p.Glu384Gly	p.Leu445Trp
6	4	M	8	Pediatrics	EVA^a	No	Yes	Congenital	70.8	Positive	p.Thr410Met	p.Tyr530His
7	5	F	5	Pediatrics	EVA^a	Yes, homogeneous	Yes	Congenital	1390	Positive	p.Arg409His	p.Arg409His
8	6	F	14	Pediatrics	EVA^a	Yes, nodular	Yes	Congenital	2200	Negative	p.Cys400Valfs^*32	p.Gln421Pro
9	7	M	24	Endo	EVA	Yes, nodular	Yes	Congenital	16	ND	p.Tyr416Pro	p.Leu445Trp
10	8	M	21	Endo	EVA	No^b	Yes^c	21 years	ND	Positive	c.1001+1G>A	p.Phe709Leufs^*12
11	8	M	23	Endo	EVA	Yes, nodular	Yes^c	23 years	ND	Positive	c.1001+1G>A	p.Phe709Leufs^*12
12	9	M	41	Endo	ND	Yes	Yes	<1 year	ND	ND	p.Tyr530His	c.2089+1G>A
13	10	M	7	Endo	EVA^a	No	Yes	Congenital	320	Negative	p.Ser28Arg	c.1615-2A>G
14	11	F	17	Endo	EVA^a	Yes, nodular	Yes^c	17 years	8650	Negative	c.1341+1G>C	c.1341+1G>C
15	12	F	29	Endo	EVA	Yes, nodular	No		91.9	Negative	p.Gly209Val	p.Glu384Gly
16	12	F	37	Endo	EVA	No^d	No		ND	ND	p.Gly209Val	p.Glu384Gly
17	12	F	33	Endo	ND	Yes, nodular	No		ND	Negative	p.Gly209Val	p.Glu384Gly
18	13	M	19	Otolaryn	EVA	Yes, nodular	Yes^c	Childhood	117.9	Negative	p.Thr416Pro	p.His723Arg
19	14	F	32	Otolaryn	EVA^a	Yes, nodular	No		391	Positive	p.Glu384Gly	p.Leu727Tyrfs^*28

Normal range for thyroglobulin: 1.5–43 ng/mL. Elevated thyroglobulin values are indicated in bold. New mutant allele is indicated in bold.

Associated cochlear dysplasia.

Nodular thyroid.

Subclinical hypothyroidism.

Papillary microcarcinoma.

Endo, endocrinology; EVA, enlargement of the vestibular aqueduct; Otolaryn, otolaryngology; ND, not done; Hypo, hypothyroidism; Tg, thyroglobulin; PDT, perchlorate discharge test.

Goiter was identified in 15 patients (79%). The age at diagnosis ranged from birth to 35 years. Goiter was nodular in all but one patient, the youngest patient at evaluation, aged five years (patient 7). Six patients underwent total thyroidectomy. The size of goiter ranged from 20.4 mL (ultrasound measurement) to 142 g (at surgical removal) in adult patients. The four patients with normal thyroid volume were aged 7, 8, 21, and 37 years at evaluation. The two oldest of these four patients had a nodular thyroid, which was associated with subclinical hypothyroidism and a positive PDT in the 21 year old (thyroid volume 14.2 mL; patient 10). The 37-year-old patient (patient 16) underwent thyroidectomy (thyroid volume 15.7 mL), and the pathological evaluation revealed a papillary microcarcinoma. She had normal thyroid function, and PDT was not performed. The seven-year-old boy (patient 13) had hypothyroidism and a negative PDT. The eight-year-old boy (patient 6) had congenital hypothyroidism and a positive PDT.

Hypothyroidism was diagnosed in 15 patients (79%). Hypothyroidism was subclinical in four patients, congenital in six patients, and diagnosed before one year of age in three patients who were born after the introduction of systematic neonatal screening in France. In two of the nine patients with early onset hypothyroidism, it was transient, and they recovered normal thyroid function at the age of 6 and 11 years (patients 1 and 2). In a seven-year-old patient (patient 13), congenital hypothyroidism was the only thyroid abnormality (no goiter, negative PDT).

PDT was positive in 10 of 16 patients (63%). A positive PDT was associated with goiter and hypothyroidism in seven patients, with goiter and euthyroidism in one patient, and with hypothyroidism but no goiter in two patients.

The serum thyroglobulin level was elevated in 11 of 12 patients tested; the values ranged from 16 to 8650 ng/mL (normal range 1.5–43 ng/mL). In one patient, serum antithyroperoxidase and antithyroglobulin antibodies were detected (1/12; patient 4).

Genetic analysis revealed two SLC26A4 mutant alleles in all 19 patients. No patient was carrying only one mutant allele. The mutations were homozygous in two families and compound heterozygous in the other 12 families. We identified one novel variant, p.Leu727Tyrfs*28.

Patients with nonsyndromic EVA (23 patients, 23 families)

The mean age was 23.4 years (range 5–53; 8 males and 15 females; Table 2). EVA was bilateral in 16/23 and unilateral in seven. In six patients, EVA was associated with cochlear dysplasia, which differed from Mondini cochlea. CT was performed in 15 patients, MRI in two, and both CT and MRI in six; the results were similar for both techniques.

Table 2.

Clinical Characteristics and Genetic Analysis of Patients with Nonsyndromic Enlargement of the Vestibular Aqueduct

										SLC26A4 genotype
No	Family	Sex	Age	Recruitment	EVA	Goiter	Hypo	Tg (ng/mL)	PDT	Allele 1	Allele 2
20	15	M	19	Otolaryn	B	No	No	18.5	Negative	p.Gly389Arg	wt
21	16	M	48	Otolaryn	B	No	No	28.4	Negative	p.Thr416Pro	wt
22	17	M	26	Otolaryn	U^a	No	No	6.9	ND	wt	wt
23	18	F	47	Otolaryn	B	No	No	76.6	Negative	p.Thr416Pro	p.Met1Thr
24	19	F	21	Otolaryn	B	No	No	24.7	ND	wt	wt
25	20	F	17	Otolaryn	B	No	No	10	Negative	wt	wt
26	21	F	10	Otolaryn	B	No	No	11.3	Negative	p.Leu597Ser	wt
27	16	M	16	Otolaryn	B	No	No	48.6	Negative	p.Asp724Gly	p.Arg409His
28	23	M	11	Otolaryn	B^a	No	No	7.88	Negative	p.Asn324Tyr	wt
29	24	F	11	Otolaryn	B	No	No	ND	ND	p.Glu29Gln	wt
30	25	M	54	Otolaryn	U	No	No	ND	ND	wt	wt
31	26	F	27	Otolaryn	B^a	No	No	26.1	Negative	wt	wt
32	27	F	20	Otolaryn	U	No	No	7.2	Negative	wt	wt
33	28	F	53	Otolaryn	U	No	No	22	Negative	wt	wt
34	29	M	8	Otolaryn	B	No	No	7.95	ND	p.Gly663Arg	wt
35	30	F	16	Otolaryn	B	No	No	10.6	Negative	wt	wt
36	31	F	17	Otolaryn	U^a	No	No	15.4	Negative	wt	wt
37	32	F	34	Otolaryn	B	No	No	1	Negative	wt	wt
38	33	F	6	Otolaryn	B	No	No	15.79	Negative	p.Ser133^*	wt
39	34	F	11	Otolaryn	B^a	No	No	23.6	Negative	wt	wt
40	35	F	9	Otolaryn	B^a	No	No	20.98	Negative	wt	wt
41	36	M	9	Otolaryn	U	No	No	26	Negative	wt	wt
42	37	F	43	Otolaryn	U	No	No	1.09	Negative	wt	wt

Normal range for thyroglobulin: 1.5–43 ng/mL. Elevated thyroglobulin values are indicated in bold. New mutant alleles are indicated in bold.

Associated cochlear dysplasia.

B, bilateral; U, unilateral; wt: wild type.

Systematic thyroid evaluation and thyroid volume were normal in all these patients. The TSH levels were within the normal range (mean value 1.71 μIU/mL). A PDT was performed in 17 of the 23 patients and was negative in all; thyroid scintigraphy was not performed in two patients, and PDT was not performed in four because the radioiodine uptake was <10%. The serum thyroglobulin levels were slightly elevated in two patients: 76 ng/mL (normal value 1.5–43 ng/mL) in a 47-year-old woman (patient 23; thyroid volume 13.8 mL, TSH level 1.21 μIU/mL, negative PDT) and 48.6 ng/mL in a 16-year-old boy (patient 27; thyroid volume 13 mL, TSH 0.792 μIU/mL, negative PDT).

SLC26A4 mutations were identified in nine patients (9/23, 39%). Mutations were biallelic in two patients (compound heterozygous) and monoallelic in seven. The two patients with biallelic mutations (p.Thr416Pro/p.Met1Thr in patient 23; p.Asp724Gly/p.Arg409His in patient 27) were the patients with elevated thyroglobulin levels. One patient with a monoallelic mutation had unilateral EVA, and another had associated cochlear dysplasia. We identified two novel variants: p.Gly389Arg and p.Gly663Arg.

Descriptions of mutations

We identified 26 different SLC26A4 mutant alleles in 28 patients (Table 3). Three of the four frequent mutations described in Caucasian populations were observed (p.Glu384Gly, p.Thr416Pro, and c.1001+1G>A), and one mutation commonly reported in Asian populations, p.His723Arg, here identified in a Caucasian boy. The mutations were spread along the gene and the protein, without any hotspot. We did not identify a preferential location for mutations in patients with PS compared with the mutations found in patients with nonsyndromic EVA. Three mutations were identified in both phenotypes: p.Arg409His, p.Thr416Pro, and p.Leu445Trp.

Table 3.

SLC26A4 Variants Detected Among Patients with Pendred Syndrome (Patients 1–19) and Nonsyndromic Enlargement of the Vestibular Aqueduct (Patients 20–38)

	Allele 1		Allele 2
Patient	Nucleotide change	Amino acid change	Nucleotide change	Amino acid change
1	c.412G>T	p.Val138Phe	c.1334T>G	p.Leu445Trp
2	c.412G>T	p.Val138Phe	c.1334T>G	p.Leu445Trp
3	c.412G>T	p.Val138Phe	c.1151A>G	p.Glu384Gly
4	c.1151A>G	p.Glu384Gly	c.1334T>G	p.Leu445Trp
5	c.1151A>G	p.Glu384Gly	c.1334T>G	p.Leu445Trp
6	c.1229C>T	p.Thr410Met	c.1588T>C	p.Tyr530His
7	c.1226G>A	p.Arg409His	c.1226G>A	p.Arg409His
8	c.1198del	p.Cys400Valfs^*32	c.1262A>C	p.Gln421Pro
9	c.1246A>C	p.Tyr416Pro	c.1334T>G	p.Leu445Trp
10	c.1001+1G>A	Splice site	c.2127del	p.Phe709Leufs^*12
11	c.1001+1G>A	Splice site	c.2127del	p.Phe709Leufs^*12
12	c.1558T>C	p.Tyr530His	c.2089+1G>A	Splice site
13	c.84C>A	p.Ser28Arg	c.1615-2A>G	Splice site
14	c.1341+1G>C	Splice site	c.1341+1G>C	Splice site
15	c.626G>T	p.Gly209Val	c.1151A>G	p.Glu384Gly
16	c.626G>T	p.Gly209Val	c.1151A>G	p.Glu384Gly
17	c.626G>T	p.Gly209Val	c.1151A>G	p.Glu384Gly
18	c.1246A>C	p.Thr416Pro	c.2168A>G	p.His723Arg
19	c.1151A>G	p.Glu384Gly	c.2174_2177dup	p.Leu727Tyrfs^*28
20	c.1165G>C	p.Gly389Arg	wt	wt
21	c.1246A>C	p.Thr416Pro	wt	wt
23	c.1246A>C	p.Thr416Pro	c.2T>C	p.Met1Thr
26	c.1790T>C	p.Leu597Ser	wt	wt
27	c.2171A>G	p.Asp724Gly	c.1226G>A	p.Arg409His
28	c.970A>T	p.Asn324Tyr	wt	wt
29	c.85G>C	p.Glu29Gln	wt	wt
34	c.1987G>A	p.Gly663Arg	wt	wt
38	c.398C>A	p.Ser133^*	wt	wt

New mutant alleles are indicated in bold.

Three variants were new: p.Leu727Tyrfs*28 (c.2174_2177dup), p.Gly389Arg (c.1165G>C), and p.Gly663Arg (c.1987G>A). None of them was detected in a screening of 100 unaffected controls, and none has been reported in dbSNP 135 or in the Exome Variant Server (EVS, http://evs.gs.washington.edu/EVS/; Table 4). p.Leu727Tyrfs*28 (c.2174_2177dup) was detected in a patient with PS. It is located in exon 19, in the putative cytosolic carboxyterminus end of the protein, a couple of codons after p.Asp724Gly (c.2171A>G), which has been associated with a complete loss of iodide transport activity (32). This frameshift starts from Leu727 and creates a premature STOP codon 27 positions downstream. The RNA produced might be a target for nonsense-mediated decay. p.Gly389Arg and p.Gly663Arg were identified in a heterozygous state as monoallelic variants in patients with nonsyndromic EVA. p.Gly389Arg is predicted to be located in the ninth extracellular loop of the protein. This variant affects a very conserved nucleotide and amino acid. The Grantham's physicochemical distance between glycine and arginine is significant (125 [0–215)]. SIFT, PolyPhen-2, and Align-GVGD software predicts the p.Gly389Arg as a pathogenic variation. The p.Gly663Arg is located in the cytosolic carboxyterminus end of the protein in the conserved STAS domain (Gly663 is a very conserved amino acid). It also causes the replacement of a neutral glycine by a charged arginine (Grantham's physicochemical distance: 125 [0–215]) and may lead to functional consequences because this domain is thought to be involved in interactions between pendrin and nucleotides (12). Bioinformatic evaluations consider this variant as a pathogenic one (PolyPhen-2: score 0.981; Align-GVGD: C65; SIFT: score 0).

Table 4.

Bioinformatic Analysis of the New SLC26A4 Variants

Nucleotide change	Amino acid change	PolyPhen-2	SIFT	Align GVGD	dbSNP ID⁻/⁻ HMD_Mutation	EVS
c.1165G>C	p.Gly389Arg	Probably damaging (score 1)	Deleterious (score 0)	C15	Unknown	Unknown
c.1987G>A	p.Gly663Arg	Probably damaging (score 0.98)	Deleterious (score 0)	C65	Unknown	Unknown
c.2174_2177dup	p.Leu727Tyrfs^*28	NA	NA	NA	Unknown	Unknown

Nucleotide and amino acid changes are described according to the HGVS nomenclature (www.hgvs.org/mutnomen/).

EVS, Exome Variant Server (http://evs.gs.washington.edu/EVS/): exome sequence variant data obtained from 13,006 chromosomes (minor allele/major allele). dbSNP ID, Single Nucleotide Polymorphism Database identifier. HMD_Mutation, Human Mutation Database identifier. Three algorithms have been used to predict the impact of missense mutations: (i) SIFT: Sorting Intolerant From Tolerant (http://blocks.fhcrc.org/sift/SIFT.html). Variants with a score >0.05 are predicted deleterious, and with a score ≥0.05 are predicted neutral. (ii) PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/). Mutations are appraised as benign, possibly damaging, or probably damaging. (iii) Align-GVGD (http://agvgd.iarc.fr/): Grantham variation (GV), Grantham deviation (GD), class C65 corresponds to most likely interferes with functional structure-based features of the substitution site. NA, Not applicable.

Discussion

The first aim of our study was to provide an accurate description of the thyroid phenotype in PS. EVA is a constant feature in PS (33). Our study confirms the need for inner ear imaging to ensure the diagnosis (34). Goiter was part of the initial description of PS but is not now considered a constant feature because it presents in about 60% to 80% of patients (8 –10,35). Goiter appeared in 79% of our patients. Two of our four patients with normal thyroid volume were children younger than 10 years. The onset of goiter in PS is variable and is often delayed until puberty or young adulthood. Even if not present by young adulthood, it is possible that a patient will develop a goiter in future years, and this is an indication for monitoring. The other two patients were young adults, whose thyroid was nodular and therefore abnormal. Hypothyroidism was common in our patients (79%) and was overt in most patients (11/19). Previous studies reported a lower frequency in the range of 30% to 50% (9,10) and predominantly subclinical hypothyroidism. The high frequency of hypothyroidism in our patients might be explained by the fact that France was an area of moderate iodine deficiency (mean urinary iodine excretion in adults 85 μg/L). Indeed, PS patients studied in countries with high iodine intake, such as Japan and Korea, are euthyroid (25,27,36,37), whereas PS patients from low iodine intake regions can exhibit congenital hypothyroidism (38). The influence of iodine deficiency on the development of goiter and hypothyroidism is not supported by two recent studies in Slc26a4 knockout mice (39,40), suggesting that other environmental, genetic, or epigenetic factors are involved in the expression of the thyroid phenotype. However, species-related differences regarding apical iodide transport in the thyrocytes or response to iodine deficiency could also potentially explain the discordant thyroid phenotypes observed in mice and men. Interestingly, hypothyroidism was transient in two siblings (patients 1 and 2, family 1), underlining the fact that other factors might compensate for defective pendrin function.

We observed congenital onset (proven in six patients, probable in three) in more than a half of our hypothyroid patients, which is an atypical observation. Congenital hypothyroidism has already been described in PS but in a smaller number of patients (6,9,10). Congenital onset of hypothyroidism is probably not explained by iodine deficiency in their mothers during pregnancy. We cannot totally exclude that they present another associated etiology of congenital hypothyroidism. We ruled out evident causes but we did not perform specific genetic testing to exclude other forms of thyroid dyshormonogenesis.

The PDT was described as the most sensitive test for diagnosing thyroid changes associated with PS, but the test was negative for 37% of our patients, challenging the role of the PDT in evaluation of suspected PS patients. Other authors used the PDT to define PS (24). Our discordant results could be explained by the use of a different methodology. Indeed, PDT is a poorly defined test. The protocol description is often incomplete in the literature, and the criteria used for its interpretation are inconsistent. The criteria for positivity vary in different studies, ranging from >10% to >30% of radioiodide accumulated at the time of perchlorate administration (10,24,41). The iodide discharge is lower in partial organification defects compared with the discharge observed in complete organification defects, which is >20%. We chose a low cutoff point of 10% based on our local experience with the PDT, in order to be as sensitive as possible, which also implies a lower specificity and a possible overlap with normal subjects. However, the exact cutoff point does not seem to be relevant. A previous study analyzed the quantitatively measured rate of iodide discharge after administration of perchlorate in a cohort of patients with EVA and showed that patients were divided into two groups: one group with a measurable organification defect, and a second group with indistinguishable perchlorate discharge values (24). We administrated perchlorate three hours after radioiodine to obtain a total uptake sufficient to assess the discharge. Iodine discharge was measured at early intervals, 15 to 60 minutes after perchlorate administration. We did not perform later measurements because timing must be short in partial organification defects in order to minimize neck counts from organic iodine (41). A rapid discharge was observed in positive cases, 15 to 30 minutes after perchlorate administration. Negative PDTs were not due to a lower iodine uptake, since the mean total iodine uptake was higher in patients with a negative PDT (33.3%) compared to the mean total iodine uptake observed in patients with a positive PDT (24.3%). Our results might also be explained by iodine deficiency because the organification defect is only partial in PS. To rule out this hypothesis, we could have performed an iodide-perchlorate test, which has a greater sensitivity than perchlorate alone for the detection of organification defects. In fact, administration of an iodide load along with the radioactive tracer increases the intrathyroid iodide pool such that the capacity of the iodinating system may be saturated more readily, and more patients could show a positive PDT (41). Moreover, in addition to pendrin, other transporters are thought to be involved in apical iodide efflux. One candidate is CLC-5, a Cl⁻/H⁺ exchanger, which is also able to transport I⁻ but less selectively compared to Cl⁻. One study showed that CLC-5 was upregulated in thyroid tissue samples of a PS patient carrying the p.Val138Phe mutation, and suggested that this upregulation could compensate for the loss of pendrin function (42). However, the mechanisms of compensation are unclear. CLC-5 might directly transport I⁻ or indirectly facilitate pendrin-mediated I⁻ efflux providing Cl⁻ as a counter-ion for pendrin exchange activity. Similarly, the cystic fibrosis transmembrane conductance regulator (CFTR) seems to be expressed in thyroid cells. However, its potential involvement in I⁻ apical efflux awaits further investigation (43).

Our study confirms that the thyroid phenotype in PS is highly variable. Hypothyroidism might occur more frequently than described previously, and it can be congenital or transient. PDT should not be considered the “gold standard” test, and is probably substantially influenced by other factors such as iodine intake. PDT negativity does not exclude the diagnosis of PS. In France, there is a systematic screening for congenital hypothyroidism, but not for hearing, in newborns. Because an early diagnosis of deafness is important for normal language development, we believe that systematic hearing tests should be performed in newborns, particularly if they present with congenital hypothyroidism.

The second aim of our study was to examine the thyroid phenotype of patients referred with nonsyndromic EVA systematically in order to try identifying subclinical thyroid dysfunction, especially in patients with SLC26A4 mutant alleles. EVA is the most frequent morphological malformation of the inner ear observed in sensorineural hearing loss (44). Although several studies have described the frequency of one or two SLC26A4 mutant alleles in patients with nonsyndromic EVA, the thyroid was not explored systematically (21 –27). In our study, 23 patients with apparently nonsyndromic EVA were referred by otolaryngologists to an endocrinologist and had an accurate thyroid evaluation that included clinical examination, biological thyroid function tests, thyroid ultrasonography, and thyroid ¹²³I scintigraphy with PDT. More than one third (39%) had one (seven patients) or two (two patients) SLC26A4 mutant alleles, but none had goiter, hypothyroidism, or a positive PDT. Hence, the vast majority of patients with biallelic SLC26A4 mutations have a thyroid phenotype and PS, but in a minority of patients, biallelic SLC26A4 mutations can also cause nonsyndromic EVA.

Our results differ from other studies that suggested a strong association between two mutant alleles and PS, defined as the identification of a positive PDT and/or goiter (24,45). However, we studied a larger number of patients with two SLC26A4 mutant alleles, namely 21 patients versus 14 in each of the two previous studies, and we performed a greater number of PDT among those patients, namely 17 patients in our study versus 7 (24) and 9 patients (45) in the two previous studies. Patient recruitment was different in our study, since we studied two groups with distinct recruitment criteria, and patients were recruited either by pediatricians, endocrinologists, and otolaryngologists. This recruitment mode is original and permitted us to examine a wider panel of affected patients, but it introduced a recruitment bias and prevented us from performing statistical analysis in the overall population. Another significant difference with these two previous studies (24,45) is that they were performed in the United States where iodine intake is higher than in France.

The absence of a specific thyroid phenotype in the two patients with biallelic mutations and nonsyndromic EVA cannot be explained by partial inactivation of pendrin. Indeed, these two patients had biallelic mutations that have been characterized functionally (p.Thr416Pro/p.Met1Thr in patient 23; p.Asp724Gly/p.Arg409His in patient 27), and they all lead to a severe impairment of pendrin function. No iodide transport activity was observed with the pendrin products encoded by p.Thr416Pro (30), since they are retained in the endoplasmic reticulum (ER) (46). The p.Met1Thr products also display an intracellular retention with no surface expression (47). The p.Asp724Gly and the p.Arg409His mutations lead to a complete loss of iodide transport activity (32,48). However, these two patients had slightly elevated thyroglobulin levels, which might reflect a slight impairment in thyroidal iodine metabolism. Seven patients had nonsyndromic EVA and monoallelic SLC26A4 mutations. We cannot exclude the possibility that the second allele of the gene was carrying a mutation, which would not have been identified if located in an intronic or regulatory sequence. Haplotype analysis using microsatellite markers could be useful to confirm the link between SLC26A4 and the pathology and follow inheritance of the two alleles. However, we could not perform a SLC26A4-STR haplotype analysis to address our hypothesis because we did not have DNA from the relatives of these seven patients. We only analyzed DNA from the parents of patients 20, 26, 28, and 38. We found no example of vertical transmission of deafness in these four patients, whereas the mutant allele was always identified in one of the unaffected parents. The pathogenic role of monoallelic SLC26A4 mutations remains unclear. Choi et al. (49) suggested that EVA could be caused by one detectable SLC26A4 mutation in combination with a second undetectable SLC26A4 mutation (encoding for pendrin products with a residual function), or with a mutation in another autosomal gene. By contrast, they suggested that nongenetic factors, complex inheritance, or etiologic heterogeneity could account for EVA in patients with no detectable SLC26A4 mutation. Another involved mutant gene could be FOXI1, which acts as an upstream regulator of pendrin in mice (50), although a study in Taiwan did not identify any mutation in that gene among 101 families with EVA or PS (51). KCNJ10, which encodes a K⁺ channel that is involved in maintaining the endocochlear potential, is another candidate. Monoallelic mutations in that gene were identified in double heterozygosity in association with monoallelic SLC26A4 mutations in patients with an EVA/PS phenotype (52). Environmental factors, such as head or acoustic trauma, might also influence the phenotypic expression.

In conclusion, our data confirm that SLC26A4 mutation screening is needed in patients exhibiting PS or nonsyndromic EVA, and that PS is related to biallelic SLC26A4 mutations. By contrast, identification of biallelic SLC26A4 mutations cannot predict the thyroid phenotype, and systematic thyroid evaluation is needed, including thyroid ultrasonography and measurement of TSH and thyroglobulin levels. Thyroid ¹²³I scintigraphy with PDT does not seem to be helpful in predicting the thyroid phenotype, at least in regions with moderate iodine deficiency. Hence, we do not perform it systematically, especially when thyroid ultrasonography and blood tests are normal. Further surveillance of thyroid status is necessary to diagnose delayed abnormalities and also to confirm the long-term persistence of hypothyroidism. We propose a new definition of PS, based on a combination of phenotypic and genotypic criteria. We suggest the combination of three findings to confirm PS: (i) sensorineural deafness with bilateral EVA; (ii) thyroid abnormality including goiter and/or hypothyroidism (congenital or of later onset, transient or permanent, overt or subclinical) and/or a positive PDT; (iii) biallelic SLC26A4 mutations. Patients with nonsyndromic EVA and biallelic SLC26A4 should be considered as not fulfilling the diagnostic criteria, and their thyroid status should be carefully monitored. Patients with monoallelic mutations have a very low risk for developing a thyroid PS phenotype. However, we cannot exclude the possibility that the second mutation has not been identified. Hence, thyroid evaluation and follow-up is also recommended in these patients. Finally, thyroid evaluation and monitoring does not appear to be necessary in patients presenting with nonsyndromic EVA and no SLC26A4 mutation. There is a need for further larger studies with rigorous statistical analysis to confirm our conclusions.

Footnotes

Acknowledgments

This work was supported by the University Hospital of Lille, France, through a Clinical Research Hospital Project (PHRC 2002R/1922). The authors thank the practitioners who referred patients to the study: Dr. C. Stuckens, Prof. J.E. Toublanc, Prof. P. Lecomte, and Prof. A. Tabarin. Many thanks also to M. d'Herbomez for performing the thyroid biological evaluations, and to Prof. X. Marchandise for performing the thyroid ¹²³I scintigraphy and PDT.

Author Disclosure Statement

The authors have nothing to disclose.

References

Fraser

. 1965. Association of congenital deafness with goiter (Pendred's syndrome): a study of 207 families. Ann Hum Genet, 28:201–249.

Kopp

, Pesce

, Solis

. 2008. Pendred syndrome and iodide transport in the thyroid. Trends Endocrinol Metab, 19:260–268.

Phelps

, Coffey

, Trembath

, Luxon

, Grossman

, Britton

, Kendall-Taylor

, Graham

, Cadge

, Stephens

, Pembrey

, Reardon

. 1998. Radiological malformations of the ear in Pendred syndrome. Clin Radiol, 53:268–273.

Luxon

, Cohen

, Coffey

, Phelps

, Britton

, Jan

, Trembath

, Reardon

. 2003. Neuro-otological findings in Pendred syndrome. Int J Audiol, 42:82–88.

Cremers

, Admiraal

, Huygen

, Bolder

, Everett

, Joosten

, Green

, van Camp

, Otten

. 1998. Progressive hearing loss, hypoplasia of the cochlea and widened vestibular aqueducts are very common features in Pendred's syndrome. Int J Pediatr Otorhinolaryngol, 45:113–123.

Coakley

, Keir

, Connelly

. 1992. The association of thyroid dyshormonogenesis and deafness (Pendred syndrome): experience of the Victorian Neonatal Thyroid Screening Programme. J Paediatr Child Health, 28:398–401.

Morgans

, Trotter

. 1958. Association of congenital deafness with goitre; the nature of the thyroid defect. Lancet, 1:607–609.

Reardon

, Coffey

, Phelps

, Luxon

, Stephens

, Kendall-Taylor

, Britton

, Grossman

, Trembath

. 1997. Pendred syndrome—100 years of underascertainment?. Quart J Med, 90:443–447.

Reardon

, Coffey

, Chowdhury

, Grossman

, Jan

, Britton

, Kendall-Taylor

, Trembath

. 1999. Prevalence, age of onset, and natural history of thyroid disease in Pendred syndrome. J Med Genet, 36:595–598.

10.

Blons

, Feldmann

, Duval

, Messaz

, Denoyelle

, Loundon

, Sergout-Allaoui

, Houang

, Duriez

, Lacombe

, Delobel

, Leman

, Catros

, Journel

, Drouin-Garraud

, Obstoy

, Toutain

, Oden

, Toublanc

, Couderc

, Petit

, Garabedian

, Marlin

. 2004. Screening of SLC26A4 (PDS) gene in Pendred's syndrome: a large spectrum of mutations in France and phenotypic heterogeneity. Clin Genet, 66:333–340.

11.

Everett

, Glaser

, Beck

, Idol

, Buchs

, Heyman

, Adawi

, Hazani

, Nassir

, Baxevanis

, Sheffield

, Green

. 1997. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet, 17:411–422.

12.

Mount

, Romero

. 2004. The SLC26 gene family of multifunctional anion exchangers. Pflugers Arch, 447:710–721.

13.

Royaux

, Suzuki

, Mori

, Katoh

, Everett

, Kohn

, Green

. 2000. Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells. Endocrinology, 141:839–845.

14.

Gillam

, Sidhaye

, Lee

, Rutishauser

, Stephan

, Kopp

. 2004. Functional characterization of pendrin in a polarized cell system. Evidence for pendrin-mediated apical iodide efflux. J Biol Chem, 279:13004–13010.

15.

Yoshida

, Hisatome

, Taniguchi

, Sasaki

, Yamamoto

, Miake

, Fukui

, Shimizu

, Okamura

, Okura

, Igawa

, Shigemasa

, Green

, Kohn

, Suzuki

. 2004. Mechanism of iodide/chloride exchange by pendrin. Endocrinology, 145:4301–4308.

16.

Everett

, Morsli

, Wu

, Green

. 1999. Expression pattern of the mouse ortholog of the Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear. Proc Natl Acad Sci USA, 96:9727–9732.

17.

Choi

, Kim

, Ito

, Lee

, Li

, Monahan

, Wen

, Wilson

, Kurima

, Saunders

, Petralia

, Wangemann

, Friedman

, Griffith

. 2011. Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition. J Clin Invest, 121:4516–4525.

18.

Royaux

, Wall

, Karniski

, Everett

, Suzuki

, Knepper

, Green

. 2001. Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci USA, 98:4221–4226.

19.

Wall

, Hassell

, Royaux

, Green

, Chang

, Shipley

, Verlander

. 2003. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol, 284:F229–F241.

20.

Kim

, Verlander

, Matthews

, Kurtz

, Shin

, Weiner

, Everett

, Green

, Nielsen

, Wall

. 2005. Intercalated cell H+/OH− transporter expression is reduced in Slc26a4 null mice. Am J Physiol Renal Physiol, 289:F1262–1272.

21.

, Everett

, Lalwani

, Desmukh

, Friedman

, Green

, Wilcox

. 1998 A mutation in PDS causes non-syndromic recessive deafness. Nat Genet, 18:215–217.

22.

Usami

, Abe

, Weston

, Shinkawa

, Van Camp

, Kimberling

. 1999. Non-syndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations. Hum Genet, 104:188–192.

23.

Campbell

, Cucci

, Prasad

, Green

, Edeal

, Galer

, Karniski

, Sheffield

, Smith

. 2001. Pendred syndrome, DFNB4, and PDS/SLC26A4 identification of eight novel mutations and possible genotype-phenotype correlations. Hum Mutat, 17:403–411.

24.

Pryor

, Madeo

, Reynolds

, Sarlis

, Arnos

, Nance

, Yang

, Zalewski

, Brewer

, Butman

, Griffith

. 2005. SLC26A4/PDS genotype-phenotype correlation in hearing loss with enlargement of the vestibular aqueduct (EVA): evidence that Pendred syndrome and non-syndromic EVA are distinct clinical and genetic entities. J Med Genet, 42:159–165.

25.

Tsukamoto

, Suzuki

, Harada

, Namba

, Abe

, Usami

. 2003. Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese. Eur J Hum Genet, 11:916–922.

26.

, Yeh

, Chen

, Hsu

. 2005. Prevalent SLC26A4 mutations in patients with enlarged vestibular aqueduct and/or Mondini dysplasia: a unique spectrum of mutations in Taiwan, including a frequent founder mutation. Laryngoscope, 115:1060–1064.

27.

Park

, Lee

, Jin

, Lee

, Go

, Jang

, Moon

, Lee

, Chun

, Lee

, Choi

, Jung

, Griffith

, Koo

. 2005. Genetic basis of hearing loss associated with enlarged vestibular aqueducts in Koreans. Clin Genet, 67:160–165.

28.

Chanoine

, Toppet

, Lagasse

, Spehl

, Delange

. 1991. Determination of thyroid volume by ultrasound from the neonatal period to late adolescence. Eur J Pediatr, 150:395–399.

29.

Coyle

, Reardon

, Herbrick

, Tsui

, Gausden

, Lee

, Coffey

, Grueters

, Grossman

, Phelps

, Luxon

, Kendall-Taylor

, Scherer

, Trembath

. 1998. Molecular analysis of the PDS gene in Pendred syndrome. Hum Mol Genet, 7:1105–1112.

30.

Scott

, Wang

, Kreman

, Andrews

, McDonald

, Bishop

, Smith

, Karniski

, Sheffield

. 2000. Functional differences of the PDS gene product are associated with phenotypic variation in patients with Pendred syndrome and non-syndromic hearing loss (DFNB4). Hum Mol Genet, 9:1709–1715.

31.

Prasad

, Kolln

, Cucci

, Trembath

, Van Camp

, Smith

. 2004. Pendred syndrome and DFNB4-mutation screening of SLC26A4 by denaturing high-performance liquid chromatography and the identification of eleven novel mutations. Am J Med Genet A, 124:1–9.

32.

Pera

, Dossena

, Rodighiero

, Gandia

, Botta

, Meyer

, Moreno

, Nofziger

, Hernandez-Chico

, Paulmichl

. 2008. Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred syndrome and nonsyndromic EVA. Proc Natl Acad Sci USA, 105:18608–18613.

33.

Reardon

, Mahoney

CFO

, Trembath

, Jan

, Phelps

. 2000. Enlarged vestibular aqueduct: a radiological marker of Pendred syndrome, and mutation of the PDS gene. Quart J Med, 93:99–104.

34.

Fugazzola

, Cerutti

, Mannavola

, Crino

, Cassio

, Gasparoni

, Vannucchi

, Beck-Peccoz

. 2002. Differential diagnosis between Pendred and pseudo-Pendred syndromes: clinical, radiologic, and molecular studies. Pediatr Res, 51:479–484.

35.

Masmoudi

, Charfedine

, Hmani

, Grati

, Ghorbel

, Elgaied-Boulila

, Drira

, Hardelin

, Ayadi

. 2000. Pendred syndrome: phenotypic variability in two families carrying the same PDS missense mutation. Am J Med Genet, 90:38–44.

36.

Park

, Shaukat

, Liu

, Hahn

, Naz

, Ghosh

, Kim

, Moon

, Abe

, Tukamoto

, Riazuddin

, Kabra

, Erdenetungalag

, Radnaabazar

, Khan

, Pandya

, Usami

, Nance

, Wilcox

, Riazuddin

, Griffith

. 2003. Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness. J Med Genet, 40:242–248.

37.

Ganaha

, Kaname

, Yanagi

, Naritomi

, Tono

, Usami

, Suzuki

. 2013. Pathogenic substitution of IVS15+5G>A in SLC26A4 in patients of Okinawa Islands with enlarged vestibular aqueduct syndrome or Pendred syndrome. BMC Medical Genetics, 14:56.

38.

Gonzalez Trevino

, Karamanoglu Arseven

, Ceballos

, Vives

, Ramirez

, Gomez

, Medeiros-Neto

, Kopp

. 2001. Clinical and molecular analysis of three Mexican families with Pendred's syndrome. Eur J Endocrinol, 144:585–593.

39.

Calebiro

, Porazzi

, Bonomi

, Lisi

, Grindati

, De Nittis

, Fugazzola

, Marino

, Botta

, Persani

. 2011. Absence of primary hypothyroidism and goiter in Slc26a4 (−/−) mice fed on low iodine diet. J Endocrinol Invest, 34:593–598.

40.

Iwata

, Yoshida

, Teranishi

, Murata

, Hayashi

, Kanou

, Griffith

, Nakashima

. 2011. Influence of dietary iodine deficiency on the thyroid gland in Slc26a4-null mutant mice. Thyroid Res, 4:10.

41.

Wolff

. 1998. Perchlorate and the thyroid gland. Pharmacol Rev, 50:89–105.

42.

Senou

, Khalifa

, Thimmesh

, Jouret

, Devuyst

, Col

, Audinot

, Lipnik

, Moreno

, Van Sande

, Dumont

, Many

, Colin

, Gerard

. 2010 A coherent organization of differentiation proteins is required to maintain an appropriate thyroid function in the Pendred thyroid. J Clin Endocrinol Metab, 95:4021–4030.

43.

Fong

. 2011. Thyroid iodide efflux: a team effort?. J Physiol, 589:5929–5939.

44.

Valvassori

, Clemis

. 1978. The large vestibular aqueduct syndrome. Laryngoscope, 88:723–728.

45.

Madeo

, Manichaikul

, Reynolds

, Sarlis

, Pryor

, Shawker

, Griffith

. 2009. Evaluation of the thyroid in patients with hearing loss and enlarged vestibular aqueducts. Arch Otolaryngol Head Neck Surg, 135:670–676.

46.

Rotman-Pikielny

, Hirscberg

, Maruvada

, Suzuki

, Royaux

, Green

, Kohn

, Lippincott-Schwartz

, Yen

. 2002. Retention of pendrin in the endoplasmic reticulum is a major mechanism for Pendred syndrome. Hum Mol Genet, 11:2625–2633.

47.

Choi

, Stewart

, Madeo

, Pryor

, Lenhard

, Kittles

, Eisenman

, Kim

, Niparko

, Thomsen

, Arnos

, Nance

, King

, Zalewski

, Brewer

, Shawker

, Reynolds

, Butman

, Karniski

, Alper

, Griffith

. 2009. Hypo-functional SLC26A4 variants associated with nonsyndromic hearing loss and enlargement of the vestibular aqueduct: genotype-phenotype correlation or coincidental polymorphisms?. Hum Mutat, 30:599–608.

48.

Gillam

, Bartolone

, Kopp

, Bevenga

. 2005. Molecular analysis of the PDS gene in a nonconsanguineous Sicilian family with Pendred's syndrome. Thyroid, 15:734–741.

49.

Choi

, Madeo

, King

, Zalewski

, Pryor

, Muskett

, Nance

, Butman

, Brewer

, Griffith

. 2009. Segregation of enlarged vestibular aqueducts in families with non-diagnostic SLC26A4 genotypes. J Med Genet, 46:856–861.

50.

Hulander

, Kiernan

, Blomqvist

, Carlsson

, Samuelsson

, Johansson

, Steel

, Enerback

. 2003. Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant mice. Development, 130:2013–2025.

51.

, Lu

, Chen

, Yeh

, Su

, Hwu

, Hsu

. 2010. Phenotypic analyses and mutation screening of the SLC26A4 and FOXI1 genes in 101 Taiwanese families with bilateral nonsyndromic enlarged vestibular aqueduct (DFNB4) or Pendred syndrome. Audiol Neurootol, 15:57–66.

52.

Yang

, Gurrola

2nd , Wu

, Chiu

, Wangemann

, Snyder

, Smith

. 2009. Mutations of KCNJ10 together with mutations of SLC26A4 cause digenic nonsyndromic hearing loss associated with enlarged vestibular aqueduct syndrome. Am J Hum Genet, 84:651–657.