Download - A novel mutation in the pendrin gene associated with Pendred's syndrome

Clinical Endocrinology (2000) 52, 279±285

279q 2000 Blackwell Science Ltd

A novel mutation in the pendrin gene associated withPendred's syndrome

Fausto Bogazzi*, Francesco Raggi*, Federica

Ultimieri*, Alberto Campomori*, Chiara Cosci*,

Stefano Berrettini², Emanuele Neri³, Roberto La

Rocca*, Giovanni Ronca¶, Enio Martino* and

Luigi Bartalena*

*Dipartimento di Endocrinologia e Metabolismo,

Ortopedia e Traumatologia, Medicina del Lavoro,

²Clinica Otorinolaringoiatrica, ³Dipartimento Immagine

and ¶Dipartimento di Scienze dell'uomo e dell'ambiente,

University of Pisa, Pisa, Italy

(Received 28 June 1999; returned for revision 7 September

1999; ®nally revised 24 September 1999; accepted 10

November 1999)

Summary

OBJECTIVE Pendred's syndrome is an autosomal

recessive disorder characterized by goitre, sensori-

neural deafness and iodide organi®cation defect. It is

one of the most frequent causes of congenital deaf-

ness, accounting for about 10% of hereditary hearing

loss. It is caused by mutations in the pendrin (PDS)

gene, a 21 exon gene located on chromosome 7. The

aim of this study was to examine an Italian family

affected with Pendred's syndrome at the molecular

level.

PATIENTS Thirteen subjects belonging to a family

from Southern Italy were evaluated for the clinical

and genetic features of Pendred's syndrome.

MEASUREMENTS Exons 2±21 of the PDS gene were

ampli®ed from peripheral leucocytes by the polymer-

ase chain reaction; mutation analysis was performed

by single strand conformation polymorphism, direct

sequencing and restriction analysis.

RESULTS The index patient had the classical triad of

the syndrome and harboured two mutations in the

PDS gene in the form of compound heterozygosity. He

was found to be heterozygous for a cytosine to ade-

nosine mutation at nucleotide 1523 in exon 13 and for

a IVS 1001� 1G ! A mutation. The former is a novel

mutation which results in a change of 508 threonine to

asparagine in the putative eleventh transmembrane

domain. The latter mutation in the donor splice site

has already been described in other patients and is

thought to lead to aberrant splicing and premature

protein truncation. Three subjects who were hetero-

zygous for one mutation had normal phenotypes. Two

subjects had sensorineural deafness and were het-

erozygous for a single mutation. Goitre was found

only in patients with Pendred's syndrome and was

absent in all other individuals, albeit residing in an

iodine-de®cient area.

CONCLUSIONS We have identi®ed a novel mutation

in the pendrin gene causing Pendred's syndrome, and

con®rm that molecular analysis is a useful tool for a

de®nitive diagnosis. This is particularly relevant in

cases such as in the subjects of our family in which

the clinical features might be misleading and other

genetics factors might be responsible for deafness.

Introduction

Pendred's syndrome is an autosomal recessive disorder

characterized by goitre, sensorineural deafness and defective

iodide organi®cation. Goitre frequently develops during child-

hood and may require surgery due to tracheal and oesophagal

compression. Impaired organi®cation of iodide is shown by

radioiodine discharge after administration of perchlorate in the

affected subjects (Morgans & Trotter, 1958). Despite the

organi®cation defect, most patients with Pendred's syndrome

are euthyroid; only a subset of patients have hypothyroidism,

usually subclinical. The sensorineural deafness is usually pre-

lingual, and frequently associated with a defect in the spiral

lamina of the cochlea which is responsible for a single common

cavity which replaces the normal three-coil con®guration of the

inner ear (Mondini, 1791). This congenital malformation,

known as Mondini's cochlea, is not speci®c for the Pendred's

syndrome since it may be absent in some affected subjects and

present in other malformations (Ormerod, 1960). Recently, the

enlargement of the vestibular aquaduct in association with a

widened endolymphatic sac and duct has been reported to be a

constant feature of Pendred's inner ear (Phelps et al., 1998).

Pendred's syndrome is thought to account for up to 10% of cases

with congenital deafness, hence having an incidence of 7´5±10 in

100 000 individuals (Marazita et al., 1993).The gene responsible

for Pendred's syndrome was mapped to chromosome 7 and

subsequently cloned (Coyle et al., 1996; Shef®eld et al., 1996;

Everett et al., 1997). Everett et al. (1997) identi®ed mutations

in the pendrin gene of affected subjects. This gene encompasses

Correspondence: Dr Fausto Bogazzi, Dipartimento di Endocrinologia,

UniversitaÁ di Pisa, Ospedale di Cisanello, Via Paradisa, 2, 56124

Pisa, Italy. Fax: � 39 50 578772.

21 exons, contains 2343 bp open reading frame, and encodes for

a 780 aminoacid protein with 11 putative transmembrane

domains. From its homology to other proteins it was thought to

be a sulphate transporter (Everett et al., 1997). However, Scott

et al. (1999) recently reported that pendrin is not capable of

transporting sulphate, but acts as an iodide-chloride pump when

cRNA was injected into Xenopus laevis oocytes.

In this study we report the clinical and genetic features of an

Italian family with Pendred's syndrome. We identi®ed a novel

mutation in the PDS gene and show that molecular analysis is

useful for de®nitive diagnosis.

Subjects and methods

Subjects

Clinical features of the family members are summarized in

Table 1. The index patient (III-2) was a 33-yr-old male with

280 F. Bogazzi et al.

q 2000 Blackwell Science Ltd, Clinical Endocrinology, 52, 279±285

Thyroid

FT4 FT3 TSH Ab Tg volume

Patient (pmol/l) (pmol/l) (mU/l) Thyroid (pmol/l) (ml)

II-1 23´1 7´7 0´03 neg 140 21

II-2 18 3´1 0´5 neg 7´5 15

II-3*

II-4 20´6 5´4 0´9 neg 44 16

II-5 19´3 4´6 0´6 neg 27 11

II-6 12´8 6 0´8 neg 40 14

III-1 12´8 5 1´1 neg 48 13

III-2 11´6 3´1 3´9 neg 3300 121

III-3 16´7 4´8 3´8 neg 1017 93

III-4²

IV-1 14´1 5 1´0 neg 18 7

IV-2 20´6 4´6 1´6 neg 25 8

IV-3 19´3 4´8 0´8 neg 21 8

IV-4 19´3 4´6 0´9 neg 16 9

Serum hormones and antibodies were determined as described in Materials and methods. All

individuals had normal thyroid hormone levels (Thyroid volume ranged 7±121 ml). Subject II-1 had

undetectable serum TSH levels due to an autonomous thyroid nodule. * Individual II-3 deceased.

²Individual III-4 refused controls.

Table 1 Thyroid function tests in the family

members

Fig. 1 Family pedigree. Symbols indicate

goitre, deafness, mutation in exon 13 and in

exon8/intron boundary. Forward slashes

identify subjects who were deceased and

therefore unavailable for clinical and genetic

study.

the classical triad of Pendred's syndrome (Fig. 1). Clinically he

was euthyroid, had congenital deafness, but normal somatic

development and a multinodular goitre. Ultrasonography

revealed a 121-ml multinodular goitre with normoechoic

pattern. His thyroid function tests con®rmed euthyroidism:

serum FT4 11´6 pmol/l, serum FT3 3´1 pmol/l, TSH 3´9 mU/l;

thyroglobulin 3300 pmol/l; anti-Tg and anti-TPO antibody were

absent. The potassium perchlorate (KClO4) discharge test was

positive with a 68% discharge of the incorporated radioiodine

2 h after the administration of KClO4.

CT scan of the inner ear showed the presence of bilateral

partial Mondini defect of the cochlea (i.e. the absence of the 3rd

apical turn of the cochlea). MRI revealed bilateral enlarged

vestibular aquaduct (6 mm) in association with a widened

endolymphatic sac and duct (Fig. 2).

Functional and morphological thyroid assessment

Thyroid function tests were performed using commercial kits:

the normal ranges were: serum-free thyroxine (FT4), 8´4±

23´2 pmol/l, triiodothyronine (FT3), 3´8±8´4 pmol/l, TSH 0´4±

3´7 mU/l, serum Tg, < 2´25±45 pmol/l, anti-Tg and serum anti-

TPO antibody, undetectable. Thyroid volume was measured by

ultrasound and calculated by the ellipsoid model: width ´length ´ thickness ´ 0´52 for each lobe. The KClO4 discharge

test was performed as follows: two hours after administration of

a tracer dose of 131-I (50 mCi), 1-g KClO4 was administered,

and the discharge was determined after 1 and 2 h. Normal

values in our Department are below 5% discharge of the

administered radioiodine dose.

CT scan and MRI of the inner ear were performed in patient

III-2 and III-3.

The study was approved by the institutional review commit-

tee, and informed consent was obtained from all subjects.

Single strand conformation polymorphism

Single strand conformation polymorphism (SSCP) was per-

formed by standard methods. Brie¯y, 15 ml of the polymerase

chain reaction (PCR) products (up to 300 bp length) were

denaturated by heat (858C for 4 min), and electrophoresed in

nondenaturating 8±10% Tris-borate-EDTA (TBE) gel. A

thermostatically controlled refrigerated circulator was used to

maintain a constant preset temperature (208C). The gel was run

at 450 V until the bromophenol blue marker reached the bottom

of the gel. Gels were stained with silver nitrate according to a

standard protocol (Ausubel et al., 1989).

DNA sequencing

DNA was extracted from peripheral leucocytes by standard

methods (Ausubel et al., 1989). Exons 2±21 of the PDS gene

were ampli®ed by PCR using speci®c intronic primers, as

described (Everett et al., 1997). The PCR products were puri-

®ed on 1% Nusieve gel and with Wizard PCR preps DNA

puri®cation system (Promega, Madison, MA, USA). Both

strands were sequenced directly after PCR ampli®cation, using

Novel mutation in Pendred's syndrome 281


Fig. 2 CT scan (right) and MRI (left) of the inner ear of the index patient. Arrows indicate the partial Mondini's defect (right) and the widened

vestibular aquaduct (left).

FS AmpliTaq DNA polymerase and ¯uoresceinate nucleotides.

An ABI Prism 310 (Perkin Elmer) apparatus was employed.

Sequence analyses were performed using Sequencing Analysis

3´0 software.

Fok I and Hinc II restriction analysis

The 1001� 1G ! A mutation (see Results) creates a Fok I

restriction site at the exon 8/intron boundary. The presence of

the mutation was con®rmed by restriction analysis with Fok I. A

636-bp PCR product was digested with Fok I and fragments

were separated on a 1´8% TBE Metaphor gel (FMC BioProducts,

ME, USA) and visualized with ethidium bromide. Digestion of

the wild type allele resulted in two fragments of 405 and 231 bp,

at variance with the mutated allele, the digested pattern of

which comprised three fragments of 405, 210 and 21 bp.

The exon 13 mutation (see Results) abolished a Hinc II

restriction site at nucleotides 1523. The 245-bp PCR product

from normal and affected subjects was resolved after Hinc II

digestion on 1´8% TBE Metaphor gel.

Results

Deafness was present in 6 individuals and was of prelingual

onset in all of them. Two individuals were compound

heterozygotes for the PDS gene mutations; two were hetero-

zygous for one mutation, and two had no mutations in the PDS

gene (see below). Goitre was found only in the two compound

heterozygous subjects, was large in both patients and required

surgery for tracheal and oesophagal compression. The KClO4

discharge test was performed in two subjects (III-2, III-3) and

was positive in both cases (68% and 62% discharge iodide,

respectively). All subjects had normal serum FT4 and FT3

levels. Serum TSH levels were slightly elevated in subject

III-2 (3´9 mU/l) and undetectable in subject II-1, who was an

unaffected male individual with an autonomous thyroid nodule.

Serum Tg levels were markedly increased in subjects III-2 and

III-3 (3300 and 1017 pmol/l, respectively) and slightly elevated

in subject II-1 (140 pmol/l), but normal in the other individuals.

CT scan and MRI was performed also in subject III-3, showing

an identical pattern to that found in patient III-2 (Fig. 2).

SSCP

SSCP was initially used to screen the index patient. Analysis

was restricted to DNA fragment < 300 bp. The exon 13 mutation

was detected using this technique (Fig. 3) and con®rmed by

sequencing analysis.

DNA sequencing and Fok I and Hinc II restriction

analysis

Sequence analysis of the PDS gene revealed that the index

patient (III-2) was a compound heterozygous for a cytosine to-

adenosine mutation at nucleotide 1523 in exon 13 and for a

1001� 1G ! A mutation at the exon/intron boundary (Fig. 4).

The mutation in exon 13 resulted in a change of 508 threonine

to asparagine in the predicted aminoacid sequence of pendrin,

located in the eleventh transmembrane domain. The second

mutation is located at the donor splice site and might lead to a

premature truncation of the protein synthesis. In one sister of

the proband (III-3) with the classical triad of the Pendred's

syndrome, DNA analysis con®rmed the same genetic muta-

tional pattern. The mother of the the proband (II-4) was

phenotypically normal; DNA analysis revealed that she was a

carrier of the exon 13 mutation. Subjects IV-1 and IV-2 were

prelingual deaf and carrier for the 1001� 1G ! A mutation

(Fig. 4). This mutation generates an additional Fok I site

downstream of the 30end of exon 8, as con®rmed by Fok I

digestion of the mutated allele (Fig. 5). The exon 13 mutation

abolished a Hinc II restriction site, as con®rmed by Hinc II



Fig. 3 Single strand conformation polymorphism of exon 13. Lane 1

refers to the index patient (III-2), lane 2 to his sister (III-3) and lane 3

to a normal subject (II-5). Arrow indicates the mutated allele.

restriction analysis (Fig. 5). Subjects IV-2 and IV-3 had normal

phenotypes and were carriers for the mutation in exon 13.

Discussion

Mutations in the PDS gene have been identi®ed in several

families affected with Pendred's syndrome, as well as in

individuals with nonsyndromic deafness (Li et al., 1998; Van

Hauwe et al., 1998; Kopp et al., 1999) (Fig. 6). Everett et al.

(1997) found 3 homozygous mutations in 5 families, van

Hauwe et al. (1998) identi®ed mutations of the PDS gene in 14

Pendred families originating from 7 countries, and Coyle et al.

(1998) found 47 of the 60 mutated alleles in 31 families. Kopp

et al. (1999) identi®ed a deletion of thymidine 279 in exon 3,

resulting in a frameshift and a premature stop codon at

aminoacid 96, in a large inbred Brazilian kindred.



Fig. 4 Sequence analysis of exon 13 and exon 8/intron of the PDS gene. Lower panel: individual homozygous for the wild type allele (right) and

heterozygous for the exon 13 mutation (left). Mutation at nucleotide 1523 in exon 13 resulted in the change of threonine at aminoacid 508 to

asparagine.Upper panel: individual homozygous for the wild type allele (right) and heterozygous for exon 8/intron mutation (left). This mutation

in the donor splice site is believed to lead to aberrant splicing and premature protein truncation.

Fig. 5 Restriction analysis of the mutated

alleles. The mutation in exon 8/intron

sequence (right panel), created an additional

FokI restriction site. The resulting restriction

pattern consisted in a 210-bp fragment

(lane 3) instead of the 235 bp observed in the

wild type allele (lane 2). Mutation in exon 13

(left panel) abolished the HincII restriction

site (lane 2).The higher molecular band

corresponds to the mutated allele. Lane 3

refers to exon 13 wild type.

We studied at the molecular level an Italian family with

Pendred's syndrome associated with a novel compound

heterozygosity. The 1001� 1G ! A mutation at the donor

splice site had already been observed in 10 families from

Northeastern England (Coyle et al., 1998): it might lead to

aberrant splicing, either by exon skipping or by the use of a

cryptic splice site. The novel mutation of C1523A in exon 13

that we described in the present paper results in a change of

threonine to asparagine in the eleventh putative transmembrane

domain of the protein.

The analysis of our family clearly shows the autosomal

recessive pattern of Pendred's syndrome transmission. Only

individuals who were compound heterozygous for the two

mutations (III-2, III-3) had the classical triad. Subjects who

were heterozygous had normal phenotype (II-4, IV-3, IV-4) or

deaf mutism (IV-1, IV-2). It is conceivable that subject II-3

(deceased) was heterozygous for the 1001� 1G ! A mutation

since both subjects III-2 and III-3 had the same compound

heterozygosity hence excluding a de novo mutation. The fact

that subjects who were carrier of the same mutation had a normal

phenotype or were deaf, suggests that a single heterozygous

mutation is not suf®cient to cause the phenotypic expression of

the syndrome; it is likely that mutations in other genes are

responsible for deafness in subject IV-1 and IV-2. However,

different functional roles of mutations in the pendrin gene, even

in the heterozygous form, cannot be excluded.

It is worth noting that the prevalence of goitre in this family

was low in spite of their origin from an iodine-de®cient area;

goitre was present only in the two affected patients supporting a

pathogenetic basis different from iodine de®ciency.

Pendrin has some degree of homology with the human DRA

(down regulated in adenoma) and DTD (diastrophic dystrophia)

proteins which are considered sulphate transporters (Everett

et al., 1997). This homology led to the hypothesis that mutations

in the PDS gene were responsible for a reduced sulphation of

thyroglobulin and hence of its iodination. The effect of pendrin

mutations on the developing inner ear was thought to be a local

form of chondrodysplasia. Recently, Scott et al., (1999) showed

that pendrin cRNA injected into Xenopus laevis oocytes acted

as an iodide±chloride transporter. This favours the possibility

that impaired anion transport into the endolymphatic aquaduct

might increase its internal pressure and lead to the damage of

hairy cells and the atrophy of auditory ®bres. Thyroidal iodide

trapping system relies on an active transport due to the Na±I

symporter, which is active in patients with Pendred's syndrome

(Vilijn & Carrasco, 1989). Thus, it seems unlikely that pendrin

contributes to thyroidal iodine uptake. It might be postulated

that pendrin plays a role in `presenting' iodide to TPO for the

organi®cation reaction which occurs at the level of the apex of

the thyrocyte. This, however, remains unproven.

Functional studies will allow a better understanding of the

physiological role of pendrin in the organi®cation reaction of

iodide and its involvement in the developing inner ear.

Further studies on patients with Pendred's syndrome will be

of great interest to establish both the real prevalence of this

disorder and the role of genetic analysis in the diagnosis of this

syndrome.

Acknowledgements

We thank Professor A. Pinchera for his continuous encourage-

ment and advice, and Dr Mameli for the sequencing analysis.



Fig. 6 Mutations in the pendrin gene.

Localization of the known mutations in the

pendrin gene was obtained from the literature

(Everett et al., 1997; Coyle et al., 1998;

Van Hauwe et al., 1998; Kopp et al., 1999)

and from the present study. Numbers identify

transmembrane domains. B, Mutations in

the coding sequence of the pendrin gene;

A, mutations in introns: their position in the

protein domains identify the position at

which protein synthesis is supposed to be

truncated.

This work was supported in part by grants from the Ministero

dell'UniversitaÁ e della Ricerca Scienti®ca e Tecnologica,

Rome, Italy (40%) and from Fondi d'Ateneo of the University

of Pisa to E.M. and L.B.

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