Introduction
Pendred`s syndrome (PS) is an autosomic recessive disease accounting for 4-10% of congenital hearing losses. It was firstly described in 1896 as the combination of deafness and goiter (1). More recently, the phenotype of PS has been better defined. The constant feature of PS is the severe/profound sensorineural hearing loss (SNHL), invariably associated with malformations of the inner ear such as the enlargement of the vestibular aqueduct (EVA), the enlargement of the endolymphatic duct and sac (EED and EES) and, in some cases a cochlear malformation known as Mondini cochlea (2). In about half of the patients goiter of different sizes and subclinical hypothyroidism are observed, whereas in the remaining cases the thyroid has a normal volume and function. In about 80% of patients a partial iodide organification defect has been documented (3). One hundred years after the recognition of PS, the disease gene (SLC26A4 or PDS) has been cloned and mapped on the long arm of chromosome 7 (4). The putative encoded protein, pendrin, belongs to a superfamily of exchangers of chloride and other anions, such as bicarbonate and formate (5). It is characterized by intracellular N-terminus and C-terminus and by 12 transmembrane domains.

Pendrin expression

In humans the highest pendrin expression has been found in the thyroid, but it is also expressed in the kidney, in the endolymphatic duct and sac of the inner ear, in the breast and in the testis (6-9). Pendrin is also expressed in the endometrium, where it seems to have a different localization during the menstrual cycle (10), and in the placenta, where the expression increases during gestation.
PDS expression is significantly different in rats and mice, where the expression at the kidney level is higher than that of the thyroid (8). This is consistent with the lack of functional and/or microscopic thyroid alteration  in the Pds KO mouse (11). Therefore, a difference between humans and rodents in the function of pendrin itself or the presence of other regulatory factors that may influence pendrin expression, can be hypothesized.

Pendrin function

• Thyroid

At the thyroid level pendrin has been found to be located at the apical membrane of the thyroid cell facing the lumen of the follicle (6, 12). Pendrin is thus believed to transport iodide from the cell to the colloid space, where iodide will be organified. An impaired function of pendrin at this level could result in a defect of iodide transport. From a clinical point of view this is predicted to result in goiter, total iodide organification defect (TIOD) and hypothyroidism. Surprisingly, the thyroid picture is very variable. Indeed, goiter is not a constant feature and can range from a slight thyroid increase to a large multinodular goiter. Furthermore, most patients are euthyroid or subclinical hypothyroid. Moreover, the perchlorate test shows only a partial organification defect (PIOD). In accordance with this “mild” thyroid phenotype, it has been hypothesized that in the absence of pendrin function, an iodide flux into the colloid space may still occur through one or more transport systems. Thus, the role of pendrin in the thyroid is still not defined. Indeed, since the first study in Xenopus oocytes and insect cells indicating that pendrin mediates chloride and iodide transport (13), other observations have been obtained. In particular, it has been shown that in mammalian cells, pendrin is able to transport iodide only at high concentrations and that the function is independent from chloride (14). Furthermore, it has been suggested that chloride concentrations in thyroid cells are too high for iodide to be transported against it (15). Recent studies from our group, are in favour of a role of pendrin in iodide transport. Indeed, by means of experiments in which chloride was substituted by iodide, a transport of both ions in the same cellular system (Hek 293) by pendrin was demonstrated, revealing a Cl-/I- exchange with a 1:1 stechiometry (16, 17).

• Kidney

Much more is known about pendrin function at the renal level. Many studies have shown that pendrin plays a critical role in bicarbonate secretion and regulation of acid-base transport (8, 18, 19). Pendrin is localized in the connecting tubule and in the collecting duct of the kidney cortex and in particular at the apical membrane of a subpopulation of intercalated cells (type B and non-A non-B) (8). These cells carry out a fine regulation of acid-base excretion through bicarbonate-transport processes (18). Experiments in mouse and rat confirm a role of pendrin in these processes. Indeed, in basal conditions, pendrin has an apical membrane localization and a bicarbonate loading leads to an increase in pendrin expression. On the contrary, an acid loading induces a reduction in pendrin expression which seems to be shifted to the cytoplasm. Bicarbonate secretion is thus supposed to be regulated by the trafficking of pendrin between apical plasma membrane and the cytoplasm (19-21). The reduction of urinary bicarbonate excretion and the development of a metabolic alkalosis found in Pds-knockout animals, further strengthens this hypothesis (8). Intercalated cells are also known to participate to chloride reabsorption. Accordingly, pendrin expression is inversely correlated to urinary chloride excretion; indeed it is increased when urinary excretion of chloride is low, and decreased when the urinary chloride excretion is high (22). In mice pendrin has also been found to be critical in the pathogenesis of mineralcorticoid-induced hypertension (23).
Despite its critical role in bicarbonate secretion, an impaired function of pendrin at this level is not associated with disturbances of renal function and particularly in the regulation of electrolytes and acid-base balance. Indeed, no renal abnormalities have been never recorded neither in patients nor in Pds-knockout animals, when studied in basal conditions. This is likely due to the redundancy of secretion-reabsorption mechanisms in the kidney or to the reduced expression of other transporters, which likely attenuates the rise in intracellular and systemic pH expected for pendrin impairment (24). However, careful studies of renal function after basic and acid loading in PS patients could reveal abnormal handling of anions transported by pendrin.

• Inner ear

In 1999 the expression of pendrin in the endolymphatic duct  (ED) and sac (ES) of the mouse was demonstrated (25). The ED is a part of the membranous labyrinth and connects the hearing (cochlea) and equilibrium (vestibular apparatus) organs to the endolymphatic sac which is located in the posterior cranial fossa. They separate 2 different compartments filled with different fluids: perilymph with a composition similar to extracellular fluids and endolymph with a potassium and protein rich, sodium low composition. It has been postulated that pendrin could be involved in the maintenance of endolymph homeostasis promoting the ionic transports (11). Recently, the importance of mitochondria-rich cells (MRC) of the ES in the transcellular transport of ions and water has been shown. Interestingly, striking similarities in ultrastructural characteristics between MRCs of the ES and renal intercalated cells have been found. In particular, the subtype B of the MRC, that are believed to function as Cl-/bicarbonate exchangers, are the most likely candidates to be affected in PS. These cells are activated after induced endolymph volume decrease and deactivated after injection of artificial endolymph and are thus believed to be specifically involved in endolymph homeostasis (26). It has been thus hypothesized that an impaired function of pendrin at this level could result in a progressive endolymph volume increase followed by the enlargement of the membranous labyrinth and of the surrounding bony structures and to a damage of the neuroepithelium leading to SNHL (Fig. 1).

Fig.1: In normal conditions, pendrin maintains the ionic exchanges between perilymph and endolymph in the membranous labyrinth which is contained in the bony structure named vestibular aqueduct. If pendrin function is lost, the endolymph volume increases resulting in the enlargement of the membranous labyrinth and of the surrounding bony structures, such as the vestibular aqueduct and the cochlea. ES: endolymphatic sac.

This mechanism is consistent with what observed at the inner ear level in the Pds KO mouse. Indeed, at ED15, the inner ears of Pds KO mouse begins to develop an enlarged ED and ES. Progressively, also the cochlea, that is normal in the heterozygous mouse, and the entire membranous labyrinth enlarge. At electron microscopy, a degeneration of the sensory cells of the inner ear is also observed, resulting in SNHL and vestibular dysfunction (11).
This endolymphatic swelling corresponds to the malformations detected radiologically in PS patients (Fig. 2). These inner ear abnormalities and the derived SNHL are a constant feature of PS.

Fig. 2: high resolution MRI section in a patient with Pendred syndrome (A1) and in a control (A2). Note the cochlea and the semicircular canals, and an enlarged endolymphatic sac that results, as expected, not visible in the normal control. The enlargement of the vestibular aqueduct at the CT scan of the petrous bone in one Pendred patient (B1) is shown, in comparison to the normal finding in a control (B2)

From genotype to phenotype

Up to date, nearly 100 different mutations of the PDS gene have been reported in the literature, spanning the entire gene, without hot spot regions (Fig. 3). However, it is worth of note that the great majority of the mutations are localized in the intracellular N- and C-terminus (http://www.medicine.uiowa.edu/pendredandbor). 

Fig. 3: Schematic representation of all PDS mutations reported to date and associated with Pendred Syndrome, with the non-syndromic hearing loss DFNB4 or with both diseases.

Interestingly, PDS mutations are associated not only to PS, but also to a non-syndromic deafness (DFNB4, enlarged vestibular aqueduct syndrome), and in some cases the same PDS mutation can be associated with PS or with DFNB4 in different families. Functional analysis of the mutations associated with PS or DFNB4 demonstrated the complete loss of chloride/iodide pendrin-mediated transport, whereas those associated with DFNB4 still allow a residual transport of both chloride and iodide, even if  at a lower level with respect to the wild-type (27). Moreover, it has been recently reported that two mutant PDS alleles are associated with PS, while a single mutated allele is frequently found in DFNB4 (28). Very recently, the intracellular trafficking of PS mutants has been studied. As above mentioned, WT pendrin is located at the plasma membrane whereas natural mutants of pendrin do not reach the plasma membrane since they are retained in the endoplasmic reticulum probably due to improper folding (29). The mutant protein has been also shown not to interfere with the arrival of WT pendrin at the plasma membrane, in accordance to the recessive mode of inheritance of the disease (30).
No genotype-phenotype correlations have been described in PS. Indeed, as described above, a great interfamilial and intrafamilial phenotypic variability has been reported (31-33). The degree of iodide deficiency could affect the clinical manifestation of the disease, but other environmental or genetic factors are likely involved.
The accurate clinical and genetic analyses on several Italian families led us to a precise characterization of the PS phenotype (3, 34). In accordance with the literature, the SNHL is invariably present, of a severe/profound degree, and it is always bilateral. The onset of deafness is congenital, bilateral and fluctuating in about 80% of cases, while develops suddenly during childhood in a minority of patients. The enlargement of the membranous labyrinth (EVA, EED and EES) is always present, whereas the Mondini cochlea has been found only in 20% of our patients. The coexistence of a vestibular disorder is rarely found, but strongly affects the quality of life of these patients. Differently from what reported in the literature, a goiter of different sizes is present in 95% of our patients and in all cases  the discharge after perchlorate ranged 35-60%, indicating a PIOD. About 80% of our cohort of patients is euthyroid and a minority has a subclinical hypothyroidism. The TSH suppressive treatment with L-thyroxine has no effect on thyroid volume reduction and the patient with the largest goiters always need thyroidectomy. No renal function alterations were never found in our patients in basal conditions. Normal menstrual cycles and at term pregnancies were recorded in female patients.

Concluding remarks

Pendrin is an interesting protein with a critical function in the inner ear. However, its role at the thyroid level is still debated and it seems not to be crucial to renal function, at least in basal conditions. The phenotype of PS is extremely variable, being the only constant feature the severe/profound hearing loss and the inner ear malformations. The differential diagnosis between PS and Pseudo-Pendred should always be done and should be based on these data. Indeed, as shown in the schematic flow-chart of Fig. 4, clinical picture such as moderate or unilateral deafness and clinical hypothyroidism argue against the diagnosis of true PS. Similarly, the absence of inner ear malformations definitely excludes PS, while the presence of these alterations strongly indicates the genetic involvement of the PDS gene. 

Fig. 4: schematic flow-chart for the differential diagnosis between Pendred and Pseudo-Pendred.