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Original Studies |
Institute of Endocrine Sciences, University of Milan (L.F., D.M., L.P., P.B.P.), Istituto Clinico Humanitas (L.F., D.M., P.B.), Istituto Auxologico Italiano IRCCS (L.P.) and Ospedale Maggiore IRCCS (P.B.P.), 20122 Milan; Departments of Internal Medicine (N.C.), Pediatric Sciences (M.M.), and Otorhinolaryngology (F.P.), Policlinico S. Matteo IRCCS, University of Pavia, 27100 Pavia; and Third Pediatric Clinic, HS Raffaele (G.W.), 20142 Milan, Italy
Address all correspondence and requests for reprints to: Paolo Beck-Peccoz, M.D., Institute of Endocrine Sciences (Pad. Granelli), Ospedale Maggiore IRCCS, Via F. Sforza 35, 20122 Milan, Italy. E-mail: paolo.beckpeccoz{at}galactica.it
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Abstract |
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Introduction |
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The onset of sensorineural hearing loss (SNHL) is generally prelingual (5), even if the course can be fluctuating and progressive (3, 12, 14), leading to various degrees of deafness. The first malformation associated with hearing loss was the so-called Mondini cochlea (8, 16), in which the cochlea, due to the deficiency of the interscalar septum, is constituted by a normal basal turn and the upper two turns forming a common cavity. However, more detailed and recent studies show that this bony malformation is a common, but not constant, feature of Pendred’s syndrome, thus drawing attention to the diagnostic value of malformations of the vestibular aqueduct (VA), endolymphatic duct (ED), and endolymphatic sac (ES) (6, 9). The VA is a bony canal that extends from the vestibule to the petrous bone. The VA enlargement is associated with fluctuating and sometimes progressive SNHL (17) and is known as the most common form of inner ear abnormality in the hearing-impaired population (18). It is present in about 80% of patients with a clinical diagnosis of Pendred’s syndrome and is often associated with other hearing losses (19, 20, 21, 22, 23), in particular the large VA syndrome (LVAS). Through this bony canal courses ED, which is a part of the membranous labyrinth and connects the sensory end organs for hearing (cochlea) and equilibrium (vestibular apparatus) to the ES. The lumen of the membranous labyrinth is filled with endolymph, a K+-rich, Na+-low, positively polarized, hyperosmotic, protein-rich fluid. The homeostasis in membranous labyrinth and the secretion and resorption of endolymph are maintained and regulated by the ED and ES cells (24). The recent application of thin section high resolution magnetic resonance imaging (MRI), showed ED and ES enlargements in 100% of patients with a clinical, but not molecularly proved, diagnosis of Pendred’s syndrome as well as with LVAS (9, 25, 26).
The disease gene, named PDS, has been mapped to chromosome 7q22-q31.1 and was fully characterized in 1997 (27). PDS complementary DNA (cDNA) contains an open reading frame of 2343 bp and encompasses 21 exons. By Northern blot analysis it has been found to be highly expressed in the thyroid, whereas weak signals have been observed in adult and fetal kidney as well as in fetal brain (27). PDS expression was also found in a human fetal cochlear cDNA library and more recently in mouse endolymphatic duct and sac (27, 28). The protein encoded by the PDS gene (pendrin) is predicted to consist of 780 amino acids (molecular mass, 86 kDa) and to contain an intracellular N-terminus, 11 transmembrane domains, and an extracellular C-terminus. The initially hypothesized role of pendrin as a sulfate transporter has not been confirmed by recent studies (29, 30). Indeed, pendrin functions as a sodium-independent transporter of chloride and iodide. In the last 2 yr, more than 30 different PDS mutations have been found in Pendred families from different countries (11, 14, 27, 31, 32, 33) and more recently also in 7 LVAS families (34, 35, 36).
The aim of the present study was to investigate the genotype-phenotype correspondence in 3 Italian families referred to our institution with a clinical diagnosis of Pendred’s syndrome. In fact, the genetic studies reported in the literature are generally lacking a full clinical and radiological evaluation (27, 31, 32); on the other hand, the specificity of the inner ear malformations is not genetically confirmed (9, 25, 26). Furthermore, as PDS mutations have been described in the literature with different nomenclatures, either starting from the first nucleotide of exon 1, which is noncoding, or from the ATG in exon 2, we unified them under the recent nomenclature recommendations (37) to reveal possible clustering and/or "hot spot" regions.
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Subjects and Methods |
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). The
affected members showed childhood deafness due to a sensorineural
defect and goiter, with positive perchlorate discharge test.
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The three brothers in family A, euthyroid at the neonatal screening, developed goiter and hypothyroidism during the first months of life. They began thyroid hormone substitutive therapy at 2–4 months of age. Patient B1 developed hypothyroidism without evidence of goiter during the childhood, and L-T4 treatment was started at 5 yr of age. In patient C1, goiter appeared soon after birth. Thyroid hormone levels constantly remained in the normal range, whereas thyroid volume progressively increased despite the administration of L-T4 therapy at TSH-suppressive doses. Total thyroidectomy was performed at the age of 16 yr due to esthetic problems. Histological examination showed a colloid goiter without signs of malignancy.
In all patients the sensorineural hearing loss was diagnosed during childhood. No patient had a history of vertigo. Patient A2 underwent bilateral myringoplasty at 12 yr of age due to chronic otitis media. The three families came from different Italian regions with moderate iodine deficiency.
Clinical studies
Thyroid examination. Serum TSH, free T4, and free T3 levels were measured with the Axsym System (Abbott Laboratories, Abbott Park, IL), thyroglobulin (Tg) was measured using Delfia human Tg (Pharmacia & Upjohn, Inc./Wallac Oy, Turku, Finland), anti-Tg and antithyroperoxidase antibodies were determined using a Liaison Kit (Byk-Sangtec Diagnostica, Dietzenbach, Germany). Thyroid ultrasonography was performed using an AU5 Esaote (Genova, Italy) instrument with a 7.5-MHz probe. A perchlorate test was carried out in all affected members; 2 h after the administration of 7.4 megabecquerels 131I, 1 g KClO4- was administered, and the discharge was measured after 1 h. The test was considered positive for a discharge rate of more than 10%.
Audiological examination. Hearing loss was assessed by pure tone audiometry. In each ear, the degree of hearing loss was classified by the pure tone average at 0.5, 1, and 2 kHz as normal [0–20 decibels (dB)], mild (20–40 dB), moderate (40–60 dB), severe (60–80 dB), or profound (>80 dB).
Radiological examination. To assess deficiency in the bony interscalar septum of the cochlea (Mondini deformity) and to evaluate the VA, high resolution computed tomography (CT) of temporal bones in the coronal and axial planes was performed (1-mm contiguous sections). The vestibular aqueduct was considered enlarged when its diameter at the midpoint between the common crus and the external aperture was 1.5 mm or more on thin CT sections (22).
High resolution, fast spin echo (FSE) T2-weighted MRI was performed (in axial and coronal planes) to study the membranous labyrinth and, in particular, the ED and ES. The size of the normal ED at its midpoint ranges from not visible to 1.4 mm (25). ES is rarely seen in normal subjects despite high quality FSE MR images, and it is considered enlarged when it is more than 2.5 mm (9, 25, 26). All of the images were photographed individually and as a three-dimensional composite.
Genomic DNA analysis. DNA was extracted from whole blood by standard methods. All 21 PDS exons were amplified using primers flanking each exon (27). Samples were subjected to 5-min denaturation at 98 C, followed by 35 3-step cycles (55 C for 1 min, 72 C for 2 min, and 94 C for 1 min) and 72 C for 10 min in a TouchDown Thermal Cycler (Hybaid, Middlesex, UK). PCR products were directly sequenced after removal of unincorporated deoxy-NTPs and primers using a GFX PCR DNA purification kit (Amersham Pharmacia Biotech, Uppsala, Sweden). An aliquot of 3–10 ng/100 bp of purified DNA and 3.2 pmol of either the forward or reverse primer were used in standard cycle sequencing reactions with ABI PRISM Big Dye terminators and run on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). The cycle-sequencing conditions consisted of 25 cycles of 96 C for 30 s, 50 C for 15 s, and 60 C for 4 min. One sequence read from each direction across the entire coding region and including intron-exon boundaries was obtained for each patient.
Analysis and revision of PDS gene mutations. All mutations reported previously have been uniformly classified according to the recent nomenclature recommendations, with the A of the ATG of the initiator Met codon denoted as +1 (37). Their localization on pendrin protein has been analyzed, and either the involvement of point mutation-prone sites (such as GC-rich regions or CpG dinucleotides) on cDNA or the presence of cluster regions has been evaluated.
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Results |
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After 2 months of L-T4
withdrawal, the three brothers of family A showed severe
hypothyroidism, with low free thyroid hormone levels and TSH values of
79 mU/L (patient A1), 86 mU/L (patient A2), and 53 mU/L (patient A3).
Serum Tg levels on and off L-T4 were
8.4 and 290 µg/L in patient A1, 550 and 11,380 µg/L in patient A2,
206 and 1616 µg/L in patient A3. Patient B1 showed, before starting
treatment, very low free T3 and free
T4 levels with high TSH levels (756 mU/L). Tg
levels were not measured. Patient C1 before thyroidectomy had normal
thyroid hormone levels, with TSH at the upper limit of normal range
(4.1 mU/L; normal range, 2.5–4.5; Table 1
). Anti-Tg and
antithyroperoxidase (anti-TPO) autoantibodies were negative in
all cases.
Patients B1 and C1 have normal intelligence, whereas the three brothers
of family A have low intelligence (according to WISC-R test; Table 1)
(38).
Ultrasound examination showed a small simple goiter (12-mL volume) in
patient A1, a multinodular goiter (25 mL) in patient A2, and a large
multinodular goiter (50 mL), with tracheal deviation in patient A3.
Patient B1 had a normal thyroid, whereas in patient C1 a large
multinodular goiter (45 mL) was documented before total thyroidectomy
(Table 1).
In all screened subjects, a positive perchlorate test was found, with a
discharge ranging from 40–70% (Table 1).
Audiological examination
The otological examination revealed mild sensorineural hearing
loss in patients A1 and A2. Patient A3 and B1 showed moderate hearing
loss, and in patient C1 the degree of sensorineural hearing loss was
severe in the right ear and profound in the left ear (Table 1).
Radiological examination
In the three members of family A and in patient B1 no radiological
malformations were found. In particular, no Mondini cochlea or enlarged
vestibular aqueduct (EVA) were seen at CT scan, and no membranous
labyrinth alterations were detected at FSE MRI (Table 1). Patient C1
had no cochlear malformations, whereas an EVA was documented
bilaterally (2.9 mm right ear, 2.6 mm left ear; Fig. 1). At FSE MRI, an enlarged ES (5 mm) and
an enlarged ED (>2 mm) were revealed in both ears (Fig. 2).
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PDS sequence analysis of the entire 1–20 exons and of the coding
portion of exon 21 did not show any mutation in the three members of
family A and in patient B1. In patient C1 a compound heterozygous
pattern was found: a single base pair deletion at nucleotide 1197 in
exon 10 (1197delT) and a single base pair insertion at nucleotide 2182
in exon 19 (2182–2183insG). In particular, the exon 10 deletion leads
to a frame shift at codon 400 (FS400) with the creation of a stop codon
at 431, and the G insertion of exon 19 results in a stop codon (Y728X;
Fig. 3). The FS400 mutation is located in
the seventh transmembrane domain of the protein, which will be deleted
for two and a half transmembrane domains and the entire COOH-terminus,
whereas the exon 19 insertion falls in the final portion of
extracellular C-terminus.
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Analysis and revision of PDS gene mutations
Until now, 35 different mutations in the PDS gene have been
described (11, 14, 27, 31, 32, 33), numbered either starting from ATG in
exon 2 or from the first nucleotide of exon 1. In Fig. 4, all mutations are uniformly classified
according to the recent nomenclature recommendations and reported with
their exon distribution, including those associated with LVAS (34, 36).
Two mutations have been found in both Pendred and LVAS families (32, 34). In Fig. 5, the position of each
mutation along the putative pendrin structure is shown. The majority of
the mutations (21 of 35, 60%) are missense mutations localized in the
3rd, 4th, and 10th transmembrane domains (n = 4); in the 3rd and
5th extracellular loops (n = 5); in the 4th intracellular loop
(n = 3); and in the COOH-terminus (n = 9). Frame-shift
mutations are a minority (14 of 35, 10 deletions and 4 insertions) and
are localized in the 1st, 7th, 9th, and 10th transmembrane domains (5
deletions and 1 insertion); in the 1st and 3rd extracellular loops (1
deletion and 1 insertion); in the 4th intracellular loop (1 deletion);
and in the COOH-terminus (3 deletions and 2 insertions). Two mutations
occur in an intronic sequence (first nucleotide of intron 7 and first
nucleotide of intron 8), thus affecting the splice donor sites. This
analysis suggests that there is no clustering in any particular domain
of the PDS gene, even if 40% of the mutations are localized in the
C-terminus of pendrin.
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Discussion |
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On the basis of these data we propose to redefine the diagnostic criteria of Pendred’s syndrome, taking into account the recent achievements concerning the expression and the putative role of pendrin. At the thyroid level, the consequence of an impaired pendrin function is the deficient organification of iodide, probably due to the lack of iodide transport across the apical membrane, where the protein has been recently immunolocalized (40). The accumulation of iodide into the thyroid cells leads to its discharge in response to perchlorate administration, rendering such a test essential to differentiate Pendred’s syndrome from nonsyndromic hearing losses. At the inner ear level, a defect in chloride transport could lead to an altered endolymph composition, resulting in damage of the neuroepithelium and enlargement of the membranous labyrinth structures with an osmotic and toxic mechanism (41). As ED and ES continue to mature until the age of 4 yr, their enlargement could lead in some cases to an alteration of the surrounding bony structures, such as the vestibular aqueduct and the cochlea. In accordance with recent clinical studies (9, 25, 26), these findings underline the higher diagnostic value of membranous labyrinth malformations compared to those of both Mondini cochlea and enlarged vestibular aqueduct, which are recorded in about 20% and 80% of Pendred patients, respectively. Therefore, the MRI study of the membranous labyrinth malformations, along with perchlorate discharge test, should be performed as primary examinations when Pendred’s syndrome is suspected. Finally, Qvortrup and co-workers (41) suggested that ES in the rat resembles a thyroid follicle with a stainable colloid substance occupying the lumen, and the chief cells of the sac functioning like the follicular cells of the thyroid, being able to synthesize, secrete, absorb, and digest proteins. Besides this intriguing similarity, the recent findings of PDS expression in both ED and ES (28) and the putative role of pendrin in the maintenance of the endolymph homeostasis, may explain why a single protein impairment can alter the function of these two very different organs.
Similar to the findings we recorded in the two families without PDS gene mutations, Coyle and co-workers (31) recently reported eight families and seven sporadic cases with a clinical diagnosis of Pendred’s syndrome based on the association of congenital SNHL and positive perchlorate test, but without PDS mutations or harboring only one PDS-mutated allele. Mutations in regulatory or intronic sequences of the PDS gene or involvement of other genes or interacting genes may explain the above findings, especially considering that the list of loci responsible for syndromic and nonsyndromic deafness continues to grow (42). Nonetheless, as Pendred’s syndrome appears to be a monogenic disease, although locus heterogeneity cannot be entirely excluded, and no malformations of the membranous labyrinth have been recorded in the two PDS-negative families described here, a diagnosis based only on clinical ground (SNHL, goiter, positive perchlorate test) is no longer tenable. The differential diagnosis should be focused on other diseases leading to positive perchlorate test, such as other iodide organification defects (TPO gene, other genes?) or Hashimoto’s thyroiditis. Although the clinical features of our PDS-negative patients at the thyroid level could be consistent with the above disorders, the congenital SNHL has never been described in patients with TPO defects or in hypothyroid patients with Hashimoto’s thyroiditis. Therefore, the diagnosis of true Pendred’s syndrome should be confined to patients presenting with the classic clinical features along with positive perchlorate test, malformations of the inner ear, and PDS mutations with a homozygous or composite heterozygous pattern.
The analysis of cDNA and protein localization for the 35 known PDS mutations unified under a unique nomenclature (37) reveals no clustering in any particular domain of the PDS gene, although the great majority of mutations (63%) are located in the second half of the protein. The exon 10 frame shift has been described, as 1421delT, in 3 Arabian families living in Northern Israel and in 1 family from Lebanon (27, 32). Our findings confirm that FS400 is a common mutation, although not restricted to the Middle East Arab population (32).
Finally, no differences in the protein localization or type of the mutation were seen among the cases reported as Pendred’s syndrome and those with a diagnosis of nonsyndromic LVAS, which lacks any thyroid involvement, as demonstrated by negative perchlorate test (34). It is worth noting that recent studies demonstrate that the enlargement of ED and ES are constant features of LVAS (26). Thus, it is tempting to speculate that PDS mutations are associated with a variable phenotypic spectrum, not necessarily comprising thyroid abnormality, but always characterized by membranous labyrinth malformations. At present, the genotype-phenotype correspondence exists only in patients with alterations in the inner ear structures. High resolution CT and MRI studies are thus mandatory to correctly diagnose Pendred’s syndrome and must be performed before engaging in PDS genetic analysis.
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Acknowledgments |
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Footnotes |
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Received November 15, 1999.
Revised March 22, 2000.
Accepted March 29, 2000.
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