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Department of Endocrinology (A.M.L.Y., S.S.G., P.H.K.E., L.K.H.K., D.H.C.K.), Singapore General Hospital, Singapore 169608; and Department of Clinical Research (Y.Z.), Singapore General Hospital, Singapore 169608
Address all correspondence and requests for reprints to: Dr. Alice M. L. Yong, Department of Medicine, Raja Isteri Pengiran Anak Saleha Hospital, Bandar Seri Begawan 2062, BRUNEI DARUSSALAM. E-mail: alicemlyong{at}yahoo.com
Abstract
We report two families in whom the index cases satisfied the
classical diagnostic criteria of Pendred’s syndrome. In family
I, two siblings were deaf, and one was normal. In family II, both
parents and two offspring were deaf. Computed tomography scans
performed in five of six of these deaf individuals showed enlarged
vestibular aqueducts in all cases, and Mondini cochlea only in family
II. Affected members in family I were compound heterozygotes inheriting
the paternal allele with a novel mutation S398del in exon 10 and a
maternal allele with two mutations IVS13+9C
G in intron 13, in
addition to H723R. In family II, the mother and one child carried both
the novel intronic IVS8–2AG and H723R mutations, whereas the father
and index case were homozygous for the IVS8–2AG mutation. A
perchlorate discharge test was positive in 50% of cases tested. In
conclusion, we concur that radiological and molecular studies should be
performed for confirmation of Pendred’s syndrome. We report, for the
first time, a Pendred’s syndrome family in which affected members had
three mutations, as well as a second family in whom the intermarriage
of two Pendred’s syndrome patients resulted in Pendred’s syndrome
offspring.
PENDRED SYNDROME (PS) is the most common cause of syndromic deafness, characterized by dyshormonogenesis, goiter, and sensorineural deafness (1). The sensorineural deafness is often prelingual in onset and associated with Mondini deformity. However, the Mondini defect is not specific for, nor invariably found in, all patients with PS (2). More recently, enlargement of vestibular aqueduct (EVA) has been recognized to be the commonest structural abnormality of the middle ear and is associated with enlargement of the endolymphatic sacs and ducts in all patients when examined by magnetic resonance imaging (2, 3).
The PS gene [pendrin (PDS) gene] was mapped (4, 5) to chromosome 7q31 and subsequently identified by positional cloning in 1997 (6). By Northern blot analysis, it has been shown to be highly expressed in the thyroid, with lower expression in adult and fetal kidneys, as well as in fetal brain (6). PDS expression has also been documented in a human fetal cochlear cDNA library (6), mouse endolymphatic duct and sac (7), and more recently in the syncytiotrophoblast cells of placenta (8), albeit at much lower levels than in the thyroid tissue. Although pendrin is closely related to a family of sulfate transport proteins, it has been recently reported that it is not capable of transporting sulfate (9) but acts as a chloride-iodide transport protein (10).
The clinical spectrum of PS varies, even within families, and using the clinical criteria alone for the diagnosis will result in underascertainment of more subtle manifestations of the syndrome (11). The availability of molecular analysis of the PDS gene will therefore be useful for making a definitive diagnosis. The aim of this study was to investigate the genotype-phenotype association in two Chinese Singaporean families in which the index case from each family showed the classical triad of PS—sensorineural deafness and goiter, with positive perchlorate discharge test (PDT).
Subjects and Methods
Subjects
We studied six individuals with sensorineural deafness and three unaffected family members from two unrelated Singaporean Chinese families with nonconsanguineous parents. The index cases from both families presented with prelingual sensorineural hearing loss and goiter. PDTs were positive. Family I (denoted A) included two affected members, AII-1 (index case, a 16-yr-old female) and AII-2 (13-yr-old female). Family II (denoted B) was unusual in that all four members of the family were deaf and mute; the index case was a 17-yr-old female (BII-1).
Clinical studies
Thyroid tests. Serum free T4 and TSH were measured with the Axsym System (Abbott Laboratories, Chicago, IL). Thyroid autoantibodies (namely TSH receptor antibody, thyroid peroxidase antibody, and Tg antibody) were determined using RIA kits (RSR, Cardiff, UK). PDT was carried out in all affected individuals (AII-1, AII-2, BII-1, and BII-2), apart from BI-1 (who was unavailable) and BI-3 (who had undergone a subtotal thyroidectomy). Two hours after the administration of 20 mCi radioactive iodine (131I) to individuals AII-1, BII-1, and BII-2 and 13 mCi 131I to AII-2, potassium perchlorate (KCLO4-) was administered orally, and the discharge was measured an hour later. KCLO4- (1 g) was administered to individuals AII-1, BII-1, and BII-2; and individual AII-2 received 400 mg KCLO4- (child dosing of 10 mg/kg). A positive test was defined as a discharge of greater than 10%.
Radiological examination. To study the presence of Mondini cochlea and EVA, high resolution computed tomography (CT) of the petrous temporal bones was performed in all affected patients, apart from BI-1.
DNA analysis
DNA sequencing. DNA was extracted from whole blood by standard methods. Exons 2–21 of the PDS gene were PCR-amplified with primers reported by Everett et al. (6). Samples were subjected to 5-min denaturation at 94 C, followed by 35 3-step cycles (94 C denaturation for 2 min, 53-59 C annealing for 2 min, and 72 C extension for 2 min) and 72 C for 10 min in a GeneAmp PCR System 9600 (Perkin-Elmer Corp. PE Applied Biosystems, Foster City, CA). PCR products were directly sequenced after the removal of unincorporated deoxynucleotide triphosphates and primers using a QIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany). An aliquot of 30–90 ng PCR product 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 377 Genetic Analyser (Perkin-Elmer Corp. PE Applied Biosystems). The cycle-sequence conditions consisted of 25 3-step cycles (96 C for 10 sec, 50 C for 5 sec, and 60 C for 4 min). One sequence reading from each direction across the entire coding region was obtained for each subject.
Mutation analysis. Numbering and nomenclature of mutations adheres to the recommendations for a nomenclature system for human gene mutation (12), in which A of the ATG of the initiator methionine codon is denoted as +1.
Results
The phenotypes and genotypes of the six individuals are presented
in Fig. 1
. We identified four
mutations in these two families, of which three are novel (Fig. 2). The
novel mutations detected were S398del in exon 10 and two intronic
mutations, IVS8–2AG and IVS13+9CG, in introns 8 and 13,
respectively.
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). The S394del mutation was inherited
from the father (AI-1). The other novel mutation identified in this
family was the IVS13+9CG (1544+9CG) in the C-terminus region
(Fig. 2b). This mutation was present on the maternal allele together
with H723R. All 3 children inherited this maternal allele, but only
those who were compound heterozygotes, inheriting the paternal S394del,
were phenotypically abnormal. The IVS13+9CG mutation was not present
in the other 5 individuals studied or in 51 controls (total 112
alleles).
The IVS8–2AG was a novel mutation detected in family II, occurring
at the acceptor splice site at nucleotide 919–2AG of intron 8 (Fig. 2c). The father (BI-1) was homozygous for the IVS8–2AG mutation,
and the mother (BI-3) also carried this mutation together with the
H723R mutation. The proband in this family (BII-1) inherited both the
IVS8–2AG and H723R mutations, whereas her affected sibling (BII-2)
was homozygous for the IVS8–2AG mutation.
The H723R missense mutation (Fig. 2d), which was present in both
the families studied, has been reported by others (13 ;
also see 15). The H723R (2168AG) mutation results
in a histidine-to-arginine substitution at codon 723, occurring in a
nonconserved area of the C-terminus of exon 19.
Deafness was of prelingual onset in all affected individuals. CT scans of all five affected individuals studied showed the presence of bilaterally EVA, and Mondini cochlea was present in all the members of family II (apart from the father, BI-1, who was not available for scanning). Both affected members from family I (AII-1 and AII-3) had a small goiter. Goiter was of a moderate size in the index case of family II (BII-1) and her mother (BI-3), who had a subtotal thyroidectomy in the past. The thyroid function test was normal in all individuals tested. Thyroid autoantibodies were all negative. PDT was positive only in the index cases of both families; with 19% and 38% discharge from individuals AII-1 and BII-1, respectively.
Discussion
Since the cloning of the PDS gene in 1997, more than 30 different mutations have been found in several Pendred families (3, 6, 13, 14, 15, 16, 17, 18, 19, 20) and in individuals with nonsyndromic deafness (14, 16, 21, 22, 23). Molecular analysis of our patients revealed 4 mutations, of which 3 were novel. The H723R mutation has been previously implicated in both PS (14) and nonsyndromic hearing loss associated with EVA (22). Its function on pendrin is unknown. However, its segregation in 3 Japanese families with EVA indicated that this mutation might be disease-causing rather than a polymorphism (22). The H723R mutation was detected in both our families, confirming that it is a common mutation and not restricted to the European or Japanese populations.
In family I, we found a novel mutation in exon 10, resulting in
1181–3delTCT at codon 394 (S394del) of transmembrane domain 9. Both
affected individuals inherited this from the father together with the
maternal allele bearing the H723R and IVS13+9CG mutations. This
represents, to our knowledge, the first reported Pendred family with
three mutations. The significance of the IVS13+9CG mutation is
unclear but does not seem to be a common polymorphism.
The IVS8–2AG was a novel mutation detected in family II, occurring
at the acceptor splice site at nucleotide 919–2AG in intron 8. The
father (BI-1) was homozygous for the IVS8–2AG mutation, but we were
unable to obtain a history of consanguineous marriage in his family.
This mutation was inherited by the index case (BII-1) with the H723R
mutation, whereas her sibling (BII-2) was homozygous for the
IVS8–2AG mutation. To date, only four mutations at the
donor/acceptor site of the intron have been reported (13, 14, 17, 18). Because the IVS8–2AG mutation changes a conserved
nucleotide of the acceptor splice site consensus sequence
(24), this mutation most probably affected the splicing of
the PDS gene. Significant PDS expression has only been documented in
the thyroid tissue, which was not available from any of the affected
members, although the mother had a subtotal thyroidectomy many years
ago. Our efforts at RT-PCR amplification of the PDS gene from
lymphoblast mRNA have been unsuccessful to date.
Family II is interesting in that it is the first documented marriage
between two individuals with PS resulting in offspring with PS. Despite
the common observation of lack of correlation between genotype and
goiter size, correlation between genotype-phenotype seemed to be well
represented at least within this family. Both father (BI-1) and
offspring (BII-2), who are deaf, without clinically palpable goiter,
share the same genotype of the homozygous IVS8–2AG mutation. The
index case (BII-1) and mother (BI-3), with both the IVS8–2AG and
H723R mutations, have moderately large goiter; and in the mother,
progressive enlargement of the goiter had led to surgical intervention.
Although we did not perform molecular analysis on the two other
affected individuals from this family (BI-2-refused and BI-4-lives
abroad, Fig. 1), it is tempting for us to assume that the same
genotype-phenotype correlation exists for the expression of goiter. If
this holds true, we can then speculate that the paternal aunt will have
the homozygous IVS8–2AG mutation, whereas the maternal aunt will
have inherited the IVS8–2AG and H723R mutations. In contrast, the
differences in genotype were not reflected at the level of middle-ear
deformity, in that all members of family II who were examined (father
not assessed) had identical CT findings. Nonetheless, the radiological
examinations in families I and II differed in that, although all
examined PS patients had EVA, only individuals from family II had the
Mondini cochlea.
Our study demonstrates, and agrees with, that of Fugazzola et al. (3) in the value of the combination of clinical, radiological, and genetic studies in the diagnosis of PS. Goiter is an inconsistent finding and, when present, often presents late in childhood or early adulthood. The PDT is useful as a diagnostic tool only when the result is positive. False-negative PDT has also been reported in 1 study, where 10 (23%) patients had evidence of mutation despite normal perchlorate analysis (25). In our study, PDT was only positive in 50% (index cases only) of cases in whom the test was performed, although PDS mutations were documented in all the affected individuals from the 2 families. It is also becoming more apparent that EVA should be considered the most likely presentation of PS.
In conclusion, the classical triad of Pendred may no longer be applicable for diagnostic purposes a century after its original description. Patients with sensorineural deafness should have middle-ear imaging; and in the presence of EVA, molecular diagnostics should probably be the next investigation of choice. Securing the diagnosis of PS is important not just for management of the condition but also to allow for genetic counseling. The recent observation that PDS gene is also expressed in the syncytiotrophoblast cells of the placenta (8) heralds the potential for prenatal genetic analysis in the future.
Footnotes
A.M.L.Y. was a fellow from the Postgraduate Training Program from the Ministry of Health, Brunei Darussalam.
Abbreviations: CT, Computed tomography; EVA, enlargement of vestibular aqueduct; KCLO4-, potassium perchlorate; PDT, perchlorate discharge test; PDS gene, pendrin gene; PS, Pendred’s syndrome.
Received November 29, 2000.
Accepted April 30, 2001.
References
This article has been cited by other articles:
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  M. Goldfeld, B. Glaser, E. Nassir, J. M. Gomori, E. Hazani, and N. Bishara CT of the Ear in Pendred Syndrome Radiology, May 1, 2005; 235(2): 537 - 540. [Abstract] [Full Text] [PDF]
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 G. Borck, C. Roth, U. Martine, G. Wildhardt, and J. Pohlenz Mutations in the PDS Gene in German Families with Pendred's Syndrome: V138F Is a Founder Mutation J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2916 - 2921. [Abstract] [Full Text] [PDF]
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 H-J Park, S Shaukat, X-Z Liu, S H Hahn, S Naz, M Ghosh, H-N Kim, S-K Moon, S Abe, K Tukamoto, et al. Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness J. Med. Genet., April 1, 2003; 40(4): 242 - 248. [Abstract] [Full Text] [PDF]
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