a novel mutation in the pendrin gene associated with pendred's syndrome
TRANSCRIPT
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
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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
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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
282 F. Bogazzi et al.
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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.
Novel mutation in Pendred's syndrome 283
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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.
284 F. Bogazzi et al.
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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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