Учебники / Genetic Hearing Loss Willems 2004

within the interval previously defined for the nonsyndromic recessive deafness locus DFNB4. Everett et al. used a positional cloning strategy to identify the PDS/SLC26A4 gene at 7q31, and demonstrated that PDS mutations underlie most, if not all, cases of Pendred syndrome (9). PDS comprises 21 exons that encode an open reading frame of 2343 base pairs. Northern blot analysis of multiple tissues detected PDS mRNA in thyroid and kidney, and cochlear expression was inferred from detection of PDS by PCR analysis of a fetal cochlear cDNA library. The polypeptide product, pendrin, is 86 kDa and contains 780 amino acids. Li and colleagues subsequently reported a large, consanguineous Indian pedigree cosegregating homozygosity for a mutant PDS allele and nonsyndromic deafness (without goiter) at the DFNB4 locus (10). Interestingly, reascertainment of the kindred originally used to map the DFNB4 locus revealed that the mutant phenotype included thyroid goiter (10).

III.PENDRIN FUNCTION

Pendrin appears to be a transmembrane protein with a long carboxy terminus, although the precise topology has yet to be experimentally determined. Whereas the computational algorithm PHDhtm predicts 11 transmembrane domains with an intracellular amino terminus and extracellular carboxy terminus (9), other programs predict 12 transmembrane domains with cytoplasmic termini (11), and at least one prediction algorithm (TMHMM 2.0) predicts only nine transmembrane domains. A cytoplasmic location of the carboxy terminus is supported by the results of immunofluorescence studies of permeabilized cells with antibodies to the carboxy terminus (12).

Pendrin was initially noted to have sequence similarity to a family of sulfate transporters (9), but subsequent electrophysiological studies of heterologously expressed pendrin have not detected sulfate transport activity (13–15). Similar heterologous expression studies have shown that it can transport iodide, chloride, formate, bicarbonate, and nitrate across plasma membranes in an energyand sodium-independent manner (13,14,16,17). Scott and Karniski extended these conclusions, demonstrating that pendrin is thus capable of mediating chloride-formate exchange, an important step in the regulation of pH by the kidney (16).

A.Pendrin Function: Thyroid

In the thyroid, antipendrin antibodies specifically bind to the apical aspect of follicular thyrocytes (11,18). Pendrin was thus proposed to mediate the

transport of iodide from folliculocytes across their apical membranes into the colloid where it is conjugated with thyroglobulin in the biosynthesis of thyroxine (8,11,19). Transport of iodide across the basolateral membrane into the folliculocyte is mediated in an energy-dependent fashion by the sodium-iodide symporter. The immunolocalization and observed iodide transport activity of pendrin strongly support a model in which PDS mutations reduce or prevent the transport of iodine into the thyroid follicle, thus inhibiting the e cient biosynthesis of thyroxine. Goiter is thought to result from compensatory hypertrophy of the thyroid follicles (4).

B.Pendrin Function: Kidney

In the kidney, a combination of immunolocalization and physiological studies utilizing isolated nephron preparations from pds knockout mice indicate that pendrin mediates bicarbonate secretion by non-alpha intercalated cells in the renal cortical collecting ducts (20–23). Although an abnormal renal phenotype has not been reported for humans or mice with PDS mutations, reduced base secretion capacity in the kidney may only be revealed with a metabolic alkali load that is so large that it exceeds the compensatory capacity of the respiratory system (20,24). It is also possible that there is functional redundancy for secretion of bicarbonate within the kidney itself. This role for base/anion exchange in the kidney raises the possibility that pendrin underlies a similar role in pH homeostasis in the inner ear (16).

C.Pendrin Function: Ear

A critical role for PDS within the auditory system was initially established by the demonstration of its specific expression in distinct regions of nonsensory inner ear epithelia thought to be important for homeostasis of endolymph (25). In situ hybridization analyses of mouse inner ears detected pds mRNA expression beginning at embryonic day 13 (E13), first in the endolymphatic duct and sac, then in the cochlea and vestibule at E15. pds mRNA is eventually expressed throughout the endolymphatic duct and sac, in the external sulcus below the spiral prominence of the cochlea, and in specific nonsensory portions of the utricle and saccule, adjacent to the maculae. This localization pattern suggested that pendrin may play a crucial role in endolymph homeostasis; the endolymphatic duct and sac are believed to be involved in endolymph resorption (25). More specifically, Kitano et al. had previously suggested that luminal chloride/bicarbonate exchange is crucial both for the production of endolymph and for the maintenance of the endolymphatic potential (26).

It is also possible that defective iodide transport by pendrin in the inner ear could cause the observed mutant PDS auditory phenotypes. Inactive thyroid hormone T4 is converted to its active form, T3, by high levels of type 2 deiodinase activity within the postnatal cochlea (27). Removal of liberated iodide ions from the inner ear might require pendrin, and PDS mutations could thus result in iodide accumulation. End product inhibition of the deiodination reaction could result in decreased production of T3, which is required for physiological and structural maturation of the organ of Corti. Iodide (or chloride) retention may lead to an osmotic imbalance with abnormal endolymphatic fluid resorption. A resorption defect with resulting osmotic imbalance might thus lead to the observed dilation of the endolymphatic system and sensory cell damage (25). PDS mutations could therefore cause sensorineural hearing loss through impairment of endolymph ionic and osmotic homeostasis, pH regulation, thyroid hormone biosynthesis, or any combination of these mechanisms.

IV. pds KNOCKOUT MOUSE

A pds / knockout mouse generated and characterized by Everett et al. has provided fascinating insights into the function of pendrin in the inner ear and the pathogenesis of hearing loss in Pendred syndrome (24). Homozygous pds / mice manifest variable degrees of vestibular dysfunction as evidenced by gait unsteadiness, circling behavior, head tilting, and abnormal performance in rotarod balance testing. Auditory brainstem response analyses demonstrated that pds / mice are deaf, whereas pds+/ heterozygotes have normal hearing.

The inner ears of pds / mice are anatomically normal until E15, at which time the endolymphatic duct and sac begin to enlarge in comparison with control mice. Shortly thereafter, the cochlea and saccule of pds / mice also become grossly enlarged and dysmorphic owing to dilatation of all of the endolymph-containing spaces. The semicircular canals become enlarged in 16% of the pds / mice. Scanning electron microscopic studies of auditory and vestibular end organs demonstrated variable, irregular inner and outer hair cell degeneration that was sometimes associated with enlarged stereocilia. Although the vestibular organs appeared normal until postnatal day 7 (P7), the maculae degenerated and the otoconia were observed to be absent or abnormally enlarged between p7 and p15, and these changes progressed as the mice aged. Interestingly, no thyroid abnormalities were detected in the

mice.

Although serum thyroid function tests and macroscopic and histological studies could not detect any abnormalities, it is possible that a subtle

iodination defect is still present. Since these phenotypic features are also incompletely penetrant in Pendred syndrome, and the auditory/vestibular phenotype is so similar to those observed in human patients, the pds knockout mouse should continue to provide an outstanding mouse model for further studies of pendrin and hearing loss in Pendred syndrome. One possible line of investigation would be analysis of endolymph pH and ionic composition in the knockout inner ear. The results of such analyses may elucidate how PDS mutations a ect endolymph pH and ionic composition. Rational pharmacological therapies might eventually be available to reverse such e ects and the progression or fluctuation of hearing loss and vestibular dysfunction that is often observed in Pendred syndrome. The pds knockout mouse could provide an animal model for preclinical testing of potential therapies.

V.PDS MUTATIONS

Mutations have been found throughout PDS; at the time of this writing, over 60 mutations have been reported (9,10,17,28–44). Thus far, mutations have been identified in nearly every coding exon. They occur in predicted transmembrane domains, extracellular and intracellular loops, and both amino and carboxy termini. Missense, splice site, and frameshift/truncation mutations have all been reported (Table 1) and occur in individuals of all ethnic backgrounds. While some mutations appear to be more common than others, no single predominant PDS mutation has been identified. An early report by Van Hauwe et al. described 14 di erent mutations in 14 unrelated families with Pendred syndrome (37). Two of these mutations, L236P and T416P, were observed in seven and five of the families, respectively, with the remainder of the identified mutations each found in only one of the 14 families. Haplotype analysis of the L236P families, which were all Western European or North American in origin, was consistent with a founder e ect. The results of a similar analysis of T416P were also consistent with a common founder. Two additional mutations, IVS8+1G>A and E384G, have also emerged as common PDS mutations among families of Western European origin.

Our recent genetic epidemiological study of PDS deafness in Asian populations has demonstrated that, while the prevalence of PDS mutations and DFNB4 deafness in Asian populations is similar to that in Western ethnic groups, the mutations are distinctly di erent (44b). Moreover, within each Asian ethnic group there is a similar degree of allelic diversity with a few predominant founder mutations. One of these founder mutations, H723R, appears to be a particularly common cause of deafness in Korea and Japan (44b).

Whenever possible, mutations are listed in format consistent with the recommendations of the Nomenclature Working Group (76): nucleotide 1 is defined here as the A of the initiator ATG codon of PDS cDNA. Those mutations marked with an asterisk (*) could not be reconciled and are listed as originally published.

Two important conclusions arise from these molecular studies: First, PDS mutations are a common worldwide cause of hereditary deafness, providing molecular confirmation of clinical epidemiological estimates in Western populations. Second, the degree of allelic diversity within and among ethnic groups will significantly reduce any utility of screening approaches for PDS mutations that include only selected exons or that screen for specific mutations, especially in ethnically heterogeneous individuals.

PDS mutations are not found in every patient with an apparent clinical diagnosis of Pendred syndrome, though. Numerous groups have reported sizable series of Pendred and nonsyndromic deafness with enlarged vestibular equeduct families analyzed with SSCP and/or sequencing analysis of all coding regions (30,32,42). In many cases, only one copy of a mutation or no mutations at all have been detected. The presence of identifiable

mutations in PDS appears to be more frequent in multiplex families; in one such series reported by Campbell et al. in 2001, PDS mutations were found in 82% (9/11) of multiplex families and in 30% (14/47) of simplex families (30). Similarly, the incidence of only one mutant allele in a ected individuals seems to be more common in isolated cases; in the Campbell study, only one mutant allele was identified in 11 of the 14 simplex cases but in only three of the nine multiplex families. It thus cannot be said with certainty that PDS mutations are responsible for all cases of Pendred syndrome. However, most linkage analyses have found inheritance consistent with a monogenic etiology (6–9,45), and many groups have postulated that those patients in whom two mutated alleles cannot be identified may have undetected intronic or regulatory region mutations (32,46). Other possible explanations include multigenic inheritance in some cases or the existence of modifying environmental factors. Campbell et al. suggested that most simplex cases of deafness with EVA are of nongenetic origin (30), but thus far no environmental etiological or contributive agent has been identified.

VI. PHENOTYPE: OTOLOGICAL

Deafness in individuals with Pendred syndrome is usually prelingual in onset, although it is not always congenital. Pure tone audiometry generally reveals downsloping or flat, severe to profound sensorineural hearing loss (4,47,48). Milder hearing impairments and other audiometric configurations have been reported. Whie bilateral hearing loss is the rule, it is sometimes not symmetrical. Many a ected individuals have a stable degree of hearing impairment, but progression and fluctuation are well documented and progression may be stepwise (often associated with minor head trauma or barotrauma) or gradual (45,47). Permanent improvement in hearing levels has not been reported.

These audiological characteristics of patients with classic Pendred syndrome are strikingly similar to those of a clinical entity that had been independently referred to as the large vestibular aqueduct syndrome (LVAS) in the otolaryngological, audiological, and radiological literature (49). Multiple studies of patients with LVAS have revealed that unilateral losses do occur, and the degree of hearing impairment does not correlate with the size of the vestibular aqueduct (49). In addition, a small 5–10-decibel conductive hearing loss component in the lower frequencies is commonly observed (50). Although the cause(s) of LVAS was (were) previously unknown, familial cases were consistent with recessive inheritance (51). Subsequent molecular studies of LVAS confirmed that it is often associated with biallelic or monoallelic PDS mutations (10), although there are also

cases with no detectable PDS mutations. It is currently not clear whether LVAS and Pendred syndrome are distinct entities or represent the extremes of a continuum of manifestations of a single disorder.

Patients with more severe inner ear malformations such as the Mondini deformity (incomplete partition of adjacent cochlear turns with variable malformations of the vestibular labyrinth) may also have increased susceptibility to leakage of inner ear perilymph into the middle ear through the oval or round windows (perilymph fistulae). Perilymph fistulae are associated with sudden hearing loss, severe rotatory vertigo, and may occasionally lead to meningitis. Vestibular dysfunction is an inconsistent and variable finding in Pendred syndrome; severity ranges from subclinical caloric hyporeflexia to severe vertiginous attacks (52,53).

VII. PHENOTYPE: GOITER

Goiter is the other clinical manifestation of Pendred syndrome (1,4). Patients are nearly always euthyroid, though mild hypothyroidism does occur and TSH levels are often at the higher end of the normal range (3–5,54). The goiter is often multinodular and occasionally requires surgical extirpation for cosmetic concerns or local mass e ects. While thyroid carcinoma has been reported in a few Pendred syndrome patients, it is not clear whether the risk is any higher than that for una ected individuals (29).

The onset of goiter typically occurs around adolescence but may occur earlier in some patients (4,55). The distinction between Pendred syndrome and nonsyndromic hearing loss (with EVA) can therefore be di cult to make during childhood. This problem is exacerbated by the subjective nature of a physical examination. While ultrasound examination with volume determinations may be helpful, normal gland size varies with age and, typically, volume determinations have not been reported in a normalized fashion. Finally, goiter due to other causes is common in some regions and populations, and can thus lead to phenocopies and the potential for misdiagnosis (39). Goiter is therefore neither a sensitive nor a specific diagnostic criterion for Pendred syndrome.

The goiter usually found in Pendred syndrome is not universal (5,8,56). PDS mutations also cause nonsyndromic deafness DFNB4 (10,57). Although the a ected members of the family originally used to define the DFNB4 region were later found to have goiter (10), Li et al. reported in 1998 on a large family with 10 a ected individuals ranging from 5 to 38 years of age (10). A ected individuals had deafness, but no goiter. Thyroid function studies were normal, but the perchlorate test was not administered so it is uncertain whether these individuals possessed a subclinical thyroid organ-