Учебники / Genetics and Auditory Disorders Keats 2002

Pure tone audiometry characteristically demonstrates a progressive bilateral symmetric sensorineural hearing loss predominantly affecting higher frequencies in 80% of Stickler’s syndrome patients (Herrmann et al. 1975; Popkin and Polomeno 1974; Ruppert et al. 1970; Spallone 1987; Stickler and Pugh 1967; Temple 1989; Zlotogora et al. 1992). Other auditory tests indicate that the primary auditory pathology resides within the cochlea (Griffith et al. 2000b; Jacobson et al. 1990; Szymko et al. 2000).

5.1.2 Genetics of Stickler Syndrome

Stickler syndrome is genetically heterogeneous and mutations have now been identified in the genes encoding the a1, a2, and a3 subunits of type XI collagen: COL11A1, COL11A2, and COL2A1 on chromosomes 6, 1, and 12, respectively (Tables 6.5, 6.6). The a3(XI) subunit is translated from an alternatively spliced transcript of COL2A1, which encodes the a1(II)

TABLE 6.6. Collagen types and their genes, chromosome map locations, and associated disorders

a The a3(XI) polypeptide is a posttranslational variant of COL2A1. Disorders associated with sensorineural hearing loss are italicized.

collagen polypeptide, a quantitatively major component of articular cartilage. In contrast, type XI collagen is a quantitatively minor fibrillar collagen originally isolated from articular cartilage, but subsequently shown to be present in other tissues (Bernard et al. 1988). It is formed by the association of its three constituent polypeptide subunits to form a triple helix, which is secreted into the extracellular matrix where their N- and C- terminal propeptides are cleaved (Prockop and Kivirikko 1995). The mature type XI collagen heterotrimers self-assemble into collagen fibrils whose diameter they are thought to regulate (Li et al. 1995).

The majority of reported Stickler syndrome mutations have been in COL2A1 (Spranger et al. 1994). These dominant mutations are predicted to cause premature chain termination and are presumed to act via a haploinsufficiency mechanism. In contrast to COL2A1, the reported Stickler syndrome mutations in COL11A1 and COL11A2 are generally missense or in-frame deletion mutations that appear to act via a dominant negative mechanism (Annunen et al. 1999; Richards et al. 1996; Sirko-Osadsa et al. 1998; Vikkula et al. 1995). The Marshall syndrome phenotype appears to be specifically associated with either splice-site mutations, or genomic deletions affecting 54 bp exons in the C-terminal half of COL11A1 (Annunen et al. 1999; Griffith et al. 1998). In contrast, other mutations in COL11A1 result in phenotypes with overlapping features of the Marshall and Stickler syndrome (Annunen et al. 1999). It is thought that these mutations of COL11A1 and COL11A2 exert their effects via a dominant negative mechanism in which the mutant polypeptides can initiate normal association with wild-type polypeptides via their C-terminal propeptide domains, but cannot complete proper assembly into a triple helix. The resulting mutant heterotrimers containing both wild-type and mutant polypeptides may then undergo abnormal posttranslational modification, secretion, assembly into fibrils, or degradation (Prockop and Kivirikko 1995).

The genetics of Stickler syndrome illustrate two important general principles: (1) genetic heterogeneity, in which a phenotype may be associated with mutations in one of several genes; (2) allelic heterogeneity of COL11A1 mutations, in which more than one phenotype (the Marshall or Stickler syndromes) may be associated with mutations in a given gene. Allelism provides insights into gene structure, function and expression by providing correlations of more than one observed mutation (genotype) with a phenotype.

5.1.3 Pathogenesis of Hearing Loss in Stickler Syndrome

Biochemical and immunohistochemical analyses have demonstrated that type II collagen is expressed in the soft tissue elements of human, rodent, and avian cochleae (Khetarpal et al., 1994; Yoo and Tomoda, 1988; Ishibe, 1989; Richardson, 1987; Thalmann, 1987), as would be expected, since Stickler syndrome mutations in COL2A1 can cause sensorineural hearing loss. Similarly, classical biochemical analyses of microdissected tissue demonstrated type XI collagen in the tectorial membrane, as well as in the basilar membrane, of the adult guinea pig (Thalmann 1993). This pattern is consistent with the pattern of Col11a1 and Col11a2 mRNA expression in embryonic and early postnatal mouse cochleae (McGuirt et al. 1999; Shpargel and Griffith 2000).

Fibrillar collagens are thought to be important for the observed tensile strength and compressibility of articular cartilage and other connective tissues in which they are expressed. It is possible that the Stickler and

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Marshall syndrome mutations in these genes may cause sensorineural hearing loss by a direct effect on the biomechanical properties of extracellular matrices in the cochlea. Alternatively, in addition to their structural roles as extracellular matrix constituents, these mutations may regulate cochlear physiology or homeostasis via other mechanisms. Distinct roles for each of the subunits are indicated by differences in their expression patterns (Lui et al. 1995; Mayne et al. 1993; Tsumaki et al. 1996; Yoshioka et al. 1995), tissue-specific alternative splicing (Oxford et al. 1995; Yoshioka et al. 1995; Zhidkova et al. 1995), and their contributions to heterotypic fibril formation in bone and the vitreous humor of the eye (Mayne et al. 1993; Niyibizi and Eyre 1989). In addition, the mammalian discoidin domain receptors (DDR1 and DDR2) with tyrosine kinase domains are activated upon receptor binding to fibrillar collagens (Shrivastava et al. 1997; Vogel et al. 1997), suggesting that extracellular collagen could directly modulate cellular responses such as growth and senescence.

5.2 Pendred Syndrome

5.2.1 Phenotype

Pendred syndrome (OMIM 274600) is an autosomal recessive disorder characterized by sensorineural hearing loss in association with thyroid gland enlargement. The thyroid dysfunction is biochemically characterized by defective organic binding of iodine (Fraser 1960; Sheffield et al. 1996). Serologic studies of thyroxin and thyrotropin levels may be normal, especially in patients tested early during the course of the disease (Cremers et al. 1998). The best method for detecting the thyroid organification defect is thought to be the perchlorate discharge test (Reardon et al. 1997), which reveals an abnormally high level of discharge of exogenously administered radioactive iodine from the thyroid gland in response to administration of perchlorate (Fraser 1960).

The hearing loss associated with Pendred syndrome is usually congenital and bilateral, and the severity may range from mild to profound (Kabakkaya et al. 1993). Vestibular function is variable in patients in whom it has been studied (Fraser 1965; Illum et al. 1972). A high proportion of affected individuals have radiologically detectable malformations of the inner ear (Johnsen et al. 1987). Johnsen et al. (1986) histologically studied six ears from five patients with Pendred syndrome and found bony cochlear changes consistent with the Mondini malformation in all preparations. The classic Mondini inner ear deformity includes a reduced number of turns of the cochlea, in comparison with the normal 2 1/2 turns, absence of the interscalar septum between turns of the cochlea, enlarged vestibules, abnormal semicircular canals, and enlarged vestibular aqueducts (Schuknecht 1980). A more recent study utilizing CT and MRI scanning concluded that the

Mondini cochlear deformity was a common, but not uniform, radiologic feature of Pendred syndrome (Phelps et al. 1998). In contrast, enlargement of the endolymphatic sac and duct in association with a large vestibular aqueduct was observed in all 20 patients examined by MRI (Phelps et al. 1998). Several different surgical therapies designed to alter the structure and physiology of the endolymphatic system have not been successful at preventing progression of sensorineural hearing loss associated with enlarged vestibular aqueducts (Jackler et al. 1988; Wilson et al. 1997).

5.2.2 Genetics of Pendred Syndrome

Pendred syndrome is transmitted in an autosomal-recessive pattern (Fraser 1965; Fraser 1960) and was mapped to chromosome 7q31 in a region known also to contain the nonsyndromic recessive deafness locus DFNB4 (Baldwin et al. 1995; Coyle et al. 1996; Sheffield et al. 1996). Everett et al. (1997) identified the Pendred syndrome gene, PDS, by a positional cloning strategy. Northern analysis demonstrated significant expression of PDS in thyroid tissue. Cochlear tissue was not included in the analysis, although it was reported that PCR analyses of a human fetal cochlear cDNA library detected the presence of PDS sequences. PDS was shown to be a novel gene whose predicted protein, pendrin, shares homology to a family of transmembrane proteins that appear to be sulfate transporters (Everett et al. 1997) (Fig. 6.6). However, the expression of human pendrin in Xenopus oocytes is associated with transport of iodine and chloride, but not sulfate (Scott et al. 1998). Similar results were obtained in a second expression system using a baculovirus vector and Sf9 host cells (Scott et al. 1998).

Everett et al. (1997) described three different PDS mutations, each of which appears to act via a loss-of-function mechanism in Pendred syndrome. Two reports published together in 1998 further expanded the spectrum of known PDS mutations in Pendred syndrome (Fig. 6.6). Van Hauwe et al. (1998) used genomic exon sequencing to identify PDS mutations in fourteen of fourteen Pendred syndrome families examined from seven different countries. The results were noteworthly for the identification of two frequent missense mutations, of which one or both were present in nine of the fourteen families. Coyle et al. (1998) used SSCP to detect PDS mutations in 56 kindreds with features suggestive of Pendred syndrome. They identified and characterized one splice site and three missense mutations that together accounted for 74% of the detected mutations. They concluded, on the basis of analyses demonstrating common linked haplotypes, that common founders were the likely source of the common mutations

(Coyle et al. 1998). The demonstration of common mutations by both groups should facilitate the molecular diagnosis of Pendred syndrome, since Pendred’s syndrome is often difficult to diagnose clinically, and is probably underascertained (Reardon et al. 1997).

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FIGURE 6.6. Postulated structure of the Pendred syndrome gene product (modified from Everett et al. 1997), and its associated mutations (Coyle et al. 1998; Cremers et al. 1998; Everett et al. 1997; Van Hauwe et al. 1998). The deduced amino acid sequence has ten predicted transmembrane domains, and is thought to transport chloride and/or iodide across cell membranes (Everett et al. 1997; Scott et al. 1998).

5.2.3 Pathogenesis of Hearing Loss in Pendred Syndrome

Establishing a direct pathogenetic link between PDS mutations and thyroid dysfunction is relatively simple. The demonstration that pendrin transports iodine across cell membranes suggests a direct mechanism for the observed inability to incorporate iodine into thyroid hormone in Pendred’s syndrome (Scott et al. 1998).

The relationship of PDS to the development and function of the cochlea is less obvious. It is possible that the cochlear abnormalities are caused by thyroid dysfunction during development of the cochlea. Congenitally hypothyroid (hyt/hyt) mice have sensorineural hearing loss associated with abnormalities of the outer and inner hair cells (O’Malley et al. 1995).

However, the cochleae of hyt/hyt mice are not malformed (O’Malley et al. 1995), suggesting that hypothyroidism itself does not necessarily cause cochlear malformations in mice. Therefore the inner ear malformations observed in Pendred syndrome might not be directly caused by hypothyroidism in utero. An alternative hypothesis is that pendrin plays a more

direct role in cochlear development. A direct role for pendrin in cochlear physiology and homeostasis is suggested by highly specific and discrete patterns of expression of its mRNA in the endolymphatic system, external sulcus of the cochlea, as well as the utricule and saccule (Everett et al. 1999).

5.2.4 PDS Mutations Cause Nonsyndromic Hearing Loss (DFNB4)

Several recent reports of PDS mutations in human subjects with nonsyndromic hearing impairment provide preliminary evidence that the observed auditory abnormalities are not a secondary effect of thyroid dysfunction. Li et al. (1998) demonstrated co-segregation of a missense mutation in PDS with profound, prelingual, nonsyndromic sensorineural hearing loss associated with isolated enlargement of the vestibular aqueducts in a large Indian pedigree. Usami et al. (1998) also has presented evidence that PDS mutations may cause nonsyndromic sensorineural hearing loss associated with enlarged vestibular aqueducts.

The PDS gene and its associated mutations illustrate several important principles of auditory genetics: First, both syndromic and nonsyndromic deafness may be caused by mutations in the same gene. It is therefore important to consider syndromic hearing loss genes as candidates for nonsyndromic hearing impairment, and vice versa (Fig. 6.1). Second, it is essential to thoroughly ascertain hearing loss families for syndromic manifestations, such as goiter. Reascertainment of the family originally used to map DFNB4 revealed the presence of goiters in affected individuals, thus establishing the diagnosis of Pendred syndrome (B. Bonne-Tamir, personal communication).

5.3 Waardenburg Syndrome

5.3.1 Phenotype

5.3.1.1 Waardenburg Syndrome Types 1 and 2

Waardenburg syndrome is characterized by autosomal-dominant transmission of sensorineural hearing loss accounting for approximately 1.4 to 2% of congenital hearing loss (Fraser 1976; Partington 1964) and patchy depigmentation affecting the skin, hair, and eyes. Neural tube defects or cleft lip/palate may also be rarely observed (Farrer et al. 1992). The two major clinical subtypes of Waardenburg syndrome are distinguished by the presence (type I; WS1; OMIM 193500) or absence (type II; WS2; OMIM 193510) of dystopia canthorum, which is a lateral displacement of the inner canthi of the eyes that can be evaluated objectively using the W biometric index (Farrer et al. 1994; Newton 1989) (Fig. 6.7). Dystopia canthorum in WS1 is highly penetrant, exceeding 90% (Arias and Mota 1978). Other features of WS1 include a wide confluent eyebrow, and a high, broad nasal root (Farrer et al. 1992). Vestibular dysfunction is common but may not be symptomatic

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FIGURE 6.7. Type I Waardenburg syndrome (from Asher and Friedman 1990). Characteristic WS1 features are present including dystopia canthorum, heterochromia irides, and a white forelock. (Reproduced from Asher and Friedman, Journal of Medical Genetics 27:617–626, Copyright 1990, with permission of the BMJ Publishing Group.)

(Marcus 1968). Iris pigmentation anomalies may include eyes of different colors, brilliant blue eyes, or two segments of different colors within a single eye. The hair, eyelashes, or eyebrows may be prematurely gray, or there may be a white forelock (Farrer et al. 1992) (Fig. 6.7). The diagnostic criteria for WS1 and WS2 have been enumerated, tabulated and reviewed (Farrer et al. 1994; Farrer et al. 1992; Liu et al. 1995; Read and Newton 1997).

There is reduced penetrance of the sensorineural hearing loss phenotype in WS1 and WS2, and within and between families the hearing loss is highly variable in severity, symmetry, onset, course, and in the observed audiometric pure-tone pattern (Fig. 6.2) (Newton 1990). Waardenburg estimated the penetrance for hearing loss at 20% (Waardenburg 1951). More recent estimates of penetrance for hearing loss are 36 to 58% for WS1, and as high as 87% in some WS2 families (Liu et al. 1995; Morell et al. 1997; Newton 1990). There are some large WS families with even higher penetrance for deafness (da-Silva 1991; Morell et al. 1992), strongly indicating the existence of modifying factors in addition to the possible effects of stochastic events

(Morell et al. 1997; Pandya et al. 1996; Read and Newton 1997).

5.3.1.2 Waardenburg Syndrome Types 3 and 4

Other Waardenburg syndrome subtypes include type III Waardenburg syndrome (WS3; OMIM 148820), also known as Klein-Waardenburg syndrome. Most WS3 cases appear to be sporadic, whereas familial cases usually demonstrate autosomal-dominant inheritance. The phenotype of WS3 is composed of the WS1 phenotype in combination with hypoplastic muscles and contractures of the upper limbs. The phenotype of type IV

Waardenburg syndrome (WS4; OMIM 277580), or Shah-Waardenburg syndrome, is a combination of the WS2 phenotype with Hirschsprung disease (Shah et al. 1981). The hallmark of Hirschsprung disease is colonic obstruction and dilation caused by a lack of autonomic nervous innervation to the colon.

5.3.2 Genetics of Waardenburg Syndrome

5.3.2.1 Mutations of PAX3 Associated with WS1, WS3 and CDHS

WS1 was mapped to chromosome 2q based upon an initial observation of a de novo chromosomal inversion on 2q in a WS1 patient (Ishikiriyama et al. 1989), followed by demonstration in two families of linkage of WS1 to the placental alkaline phosphatase locus on 2q (Asher et al. 1991; Foy et al. 1990). As predicted by Asher and Friedman (1990), comparison of the map position of WS1 with the syntenic mouse region suggested a possible mouse homologue, the Splotch mutation (Foy et al. 1990). This hypothesis was confirmed by several groups demonstrated PAX3 and Pax3 mutations in WS1 patients and the Splotch mouse, respectively (Baldwin et al. 1992; Epstein et al. 1991; Morell et al. 1992; Tassabehji et al. 1992). PAX3 (originally called HuP2) contains 10 exons encoding a transcription factor with two DNA-binding domains, a paired-domain and a paired-type homeodomain (Barber et al. 1999). Expression studies in the developing mouse demonstrate concentrated Pax3 expression in the neural crest, as well as neural crest-derived structures (Goulding et al. 1991). The neural crest contributes to glial cells in the spiral ganglion and auditory nerve, as well as the melanocytes of the stria vascularis. Mouse Pax3 is known to be expressed in developing limb buds (Bober et al. 1994), as predicted by the upper limb abnormalities observed in WS3.

Most, if not all, cases of WS1 map to the PAX3 locus on chromosome 2q3. Over 50 different mutations have been described, and different families usually have different mutations (DeStefano et al. 1998). With the likely exception of missense mutations of codon 47 discussed below, most premature-termination and amino-acid-substitution mutations are predicted to cause loss-of-function and, therefore, act via haploinsufficiency. Read and Newton recently reviewed Waardenburg syndrome and favored a protein-dosage hypothesis to explain the reduced penetrance and variable expressivity of the WS1/WS3 phenotypes (Read and Newton 1997).

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They postulated that effective levels of PAX3 protein are dependent upon the PAX3 genotype in combination with variations in the host cellular responses to PAX3. Any reduction of PAX3 dosage would result in dystopia canthorum, whereas further decreases would cause, in progression, pigmentation abnormalities, limb abnormalities as seen in WS3 patients, and neural tube defects (Read and Newton 1997). This mechanism probably does not fully account for the deafness phenotype, since there is evidence that other genes, perhaps PAX3 target genes, modify its expression (Morell et al. 1997).

Most WS3 individuals are heterozygous for PAX3 mutations (Sheffer and Zlotogora 1992). A consanguineous marriage between two WS1 individuals heterozygous for a PAX3 missense mutation, S84F, produced a deaf child homozygous for S84F who had dystopia canthorum, partial albinism, and severe upper-limb defects characteristic of WS3 (Zlotogora et al. 1995). Another patient with a phenotype similar to that of WS3 had an interstitial deletion, del(2)(q35–q36), that included PAX3 and COL4A3 (Pasteris et al. 1993). In a well studied WS3 family with four affected patients (Goodman et al. 1982; Sheffer and Zlotogora 1992), Hoth and coworkers (1993) identified an asparagine-to-histidine substitution (N47H) within the paired DNA binding domain of PAX3.

Craniofacial-Deafness-Hand-Syndrome (CDHS, MIM 122880) is a syndrome that resembles the WS3 phenotype. In one family, CDHS is characterized by profound sensorineural hearing loss, absence or hypoplasia of the nasal bones, hypoplastic maxilla, small and short nose with thin nares, limited wrist mobility, short palpebral fissures, ulnar deviation of the fingers, and hypertelorism (Sommer et al. 1983). Absence or hypolasia of nasal bones was not observed in the WS3 family previously described (J. Zlotogora, personal communication). In the CDHS family, a missense mutation in codon 47 of PAX3, N47K, was identified (Asher et al. 1996). If the N47H mutation associated with WS3 and the N47K mutation in the CDHS family are both loss-of-function mutations, the phenotypic distinction between WS3 and CDHS, including nasal bone hypoplasia in CDHS, must be due to different alleles of modifying genes in these families. An alternative explanation is that not all mutations of PAX3 are null alleles, and the phenotypic differences between CDHS and WS3 may be due to altered PAX3 transcriptional properties as a consequence of the different substitutions at amino acid residue 47. N47H and N47K could differentially alter the interaction between the PAX3 protein and a subset of its downstream target genes. This latter hypothesis may be tested when more of the PAX3 target genes are identified.

5.3.2.2 WS2, WS2 with AROA and Tietz–Smith Syndrome

The WS2 phenotype in one kindred was mapped to chromosome 3q12–p14 by Hughes et al. (Hughes et al. 1994). This was known to be the location of the human orthologue, MITF, of the mouse microphthalmia gene mi