Учебники / Genetic Hearing Loss Willems 2004

WS3 (Klein-Waardenburg syndrome) has the features of WS1, including dystopia, plus limb abnormalities. There are two very di erent sets of cases. It was recently demonstrated (12) that Waardenburg was correct in his original hypothesis about Klein’s patient: she is homozygous for WS1 (in fact, a compound heterozygote for two PAX3 mutations, G99S/R270C), although the family history is still unresolved. Two other proven homozygotes have been reported. One (13) closely resembles Klein’s patient; the other (14; our unpublished data) was a grossly abnormal fetus, conceived by incest between brother and sister with WS1 and aborted because of exencephaly detected on scan. Apart from these three cases, all other reported ‘‘WS3’’ patients had a much milder phenotype. Several had relatives with typical WS1, and some were shown to be heterozygous for PAX3 mutations typical of WS1. Thus these mild ‘‘WS3’’ patients simply represent part of the range of expression of WS1, and do not merit a separate label. Conversely, they suggest that there is always a small risk of such relatively mild limb problems in any WS1 family.

For WS4 the label Shah-Waardenburg syndrome is probably best discarded in favor of the more descriptive ‘‘Waardenburg-Hirschsprung syndrome.’’ Most cases described do not have dystopia canthorum, although there seems to be no a priori reason why some patients might not have a disturbance of development a ecting both the precursors of the enteric ganglia and the cell lineages a ected by WS1 mutations. The range of phenotypes to be expected can only be discussed once the underlying genetic defect has been defined.

IV. FORMAL GENETICS OF WAARDENBURG SYNDROME

WS1 and WS2 are autosomal dominant conditions with very variable expression. For WS1 the penetrance is very high (98–99%) because dystopia is a near-universal feature. For WS2, there are some families where penetrance is high, and a large penumbra of families with a much more vague pattern of hearing loss and/or minor pigmentary anomalies. Whether these latter families should be described as WS2 is questionable. Minor disturbances of melanocyte function (whether due to defective migration from the embryonic neural crest or a failure of proliferation or survival) are common in the general population, and these may occur as low-penetrance dominant phenotypes in families. WS2 is unquestionably genetically heterogeneous. Mutations in the only gene defined to date, MITF (see below), are largely confined to families with a pronounced dominant history, but operationally, we have been unable to define clinical criteria that separate all MITF families from the ‘‘penumbra’’ families.

WS3, as described above, is a purely clinical description of a set of WS1 homozygotes or heterozygotes, and is not a genetic entity. WS4 includes several di erent genetic conditions. Some, caused by mutations in endothelin 3 and its receptor, are recessive; others, caused by mutations in SOX10, are dominant (but often new mutations), while yet others are at present undefined. Occasional patients homozygous for mutations in EDNRB or EDN3 have pigmentary features but not Hirschsprung disease, and are describable clinically as WS2.

V.IDENTIFICATION OF GENES UNDERLYING WAARDENBURG SYNDROME

Five genes, PAX3, MITF, EDN3, EDNRB, and SOX10, have been implicated in Waardenburg syndrome, and others remain to be defined. The relation of genotypes to phenotypes is summarized in Table 3.

Table 3 Phenotypes and Genotypes in Human Auditory-Pigmentary Syndromes

A.Identification of PAX3

Linkage analysis mapped a WS1 locus to distal 2q in 1990 (15,16). This region shows strongly conserved synteny with the distal part of mouse chromosome 1. A mouse mutant, Splotch, mapped to the same area. Sp heterozygous mice have a white belly spot, while homozygotes die with lethal neural tube defects (17) or (it was later discovered) conotruncal heart defects (18). Splotch was not an obvious candidate for a WS1 homolog because the heterozygous mice had no apparent hearing defect [confirmed in later detailed work (19)], while homozygotes had inner ear malformations, placing Splotch in the morphogenetic rather than cochleosaccular category in Steel’s classification of hearing defects (20). However, an interesting candidate gene for a neurocristopathy mapped to the approximate candidate region in the mouse. This was Pax-3, a gene encoding a transcription factor expressed in the embryonic neural crest. Part of the likely human homolog of Pax-3 had been cloned by Burri et al. (21) and named HuP2. Simultaneous analysis of mouse and human DNA revealed mutations in Pax-3 in Sp and in HuP2, now renamed PAX3, in WS1 patients (22–24).

B.Identification of MITF

A promising mouse model for WS2, the microphthalmia mouse (25), mapped to mouse chromosome 6, but synteny is poorly conserved between mouse and human in this region, and attempts to use the mouse map to predict the map location of human WS2 genes were unsuccessful. A whole genome search in a large WS2 family mapped the locus to the proximal short arm of chromosome 3, at 3p14-p21 (26). Eventually mi was cloned, and the human homolog was then identified and physically mapped (27). Since the human gene, MITF, mapped to precisely the location on 3p defined by our linkage studies, MITF became a strong candidate for this WS2 gene, and mutations were soon identified (28).

C.Identification of EDNRB and EDN3

Disruption of Ednrb, encoding the endothelin receptor B, in mice unexpectedly identified that gene as the cause of a much-studied WS4 model, piebaldlethal (sl) (29). A variable Waardenburg-Hirschsprung phenotype had been mapped to human chromosome 13 in a very large Mennonite family, and a number of other case reports also suggested the presence on chromosome 13 of a gene implicated in Waardenburg-Hirschsprung disease (30). The human EDNRB gene mapped within the chromosome 13 WS-HSCR candidate

region, and so was tested for mutations. These were soon detected in the Mennonite family, and subsequently in other WS4 families (31,32). The ligand of the ENDRB receptor is endothelin 3, encoded by the EDN3 gene located on chromosome 20. In the mouse, disruption of Edn3 produced another WS-HSCR phenotype, lethal-spotting (ls), and homozygous EDN3 mutations were identified in a few WS4 patients (33,34).

D.Identification of SOX10

A third mouse model of WS-HSCR is Dominant megacolon (Dom). The gene underlying this phenotype was identified by positional cloning as Sox10 (35,36). Waardenburg, Hirschsprung, and Waardenburg-Hirschsprung patients were tested for mutations in the human homolog, SOX10, and these were found in a small number of WS-HSCR patients (37). SOX10 is located on human chromosome 22q13, but because of the severity of the phenotype, most cases are new mutations, and linkage analysis played no part in identifying the human gene.

VI. MOLECULAR PATHOLOGY OF

WAARDENBURG SYNDROME

PAX3, MITF, and SOX10 all encode transcription factors. In common with many other transcription factors, the genes are dosage sensitive—a half dose of the gene product is not enough for completely normal development (haploinsu ciency). Conditions caused by haploinsu ciency are typically very variable, even within families, and Waardenburg syndrome is typical in this respect. Presumably there is a fairly fine balance within the cell, and the final outcome will reflect both variations in the interacting partners and chance events.

PAX3 is a member of the paired box family of genes, which comprises nine genes, PAX1–PAX9, in mice and humans. Their common feature is a paired box, encoding a 128or 129-amino-acid bipartite DNA-binding domain first characterized in the Drosophila paired gene (38). The PAX3, -4, -6, and -7 genes additionally encode a 60-amino-acid homeodomain. Thus the PAX3 protein contains three DNA-binding elements, the N-terminal and C-terminal parts of the paired domain, and the paired-type homeodomain. Additionally there is a typical serine-threonine-rich transactivation domain toward the C-terminal part of the protein, and a conserved octapeptide, HSIDGILS, of unknown function located between the paired and homeo domains. The three DNA-binding elements interact, allowing a rich and subtle repertoire of interactions that are far from fully understood. Addi-

tionally there must be protein-protein interactions, which may involve the homeodomain and octapeptide as well as the transactivation domain.

The PAX3 gene comprises 10 exons, covering almost 100 kb of genomic DNA at chromosome 2q35. Exons 2–4 contain the paired box, and exons 5 and 6 the homeobox. Alternative splicing at the 3V end produces isoforms terminating in exon 8, 9, or 10, and other forms have been reported that terminate after exon 4. It is not known whether any of these di erences are functionally important. Alternative splice acceptors at the start of exon 3 produces forms with (+Q) or without ( Q) glutamine at amino acid 108. These variants show di erential patterns of transcriptional activation in an in vitro assay (39). PAX3 is expressed, as expected, in the embryonic neural crest and in some of the populations of cells migrating out of this tissue.

As is usually the case with transcription factors, identifying the downstream targets has been di cult. The DNA-binding sequences are not tightly enough specified to allow database searching to define target genes, and experimental approaches have an uncertain relation to the reality in the cell. An ambitious attempt to define targets by an in vitro cyclical DNA amplification and selection method (40) has produced a long list of possible targets, which will need painstaking validation. Known targets include MITF (41), MET (42), RET (43), and (perhaps indirectly) MyoD and/or Myf5 (44). Each of these helps explain features of Waardenburg syndrome. Control of MITF by PAX3 explains why the melanocyte features of WS1 and WS2 are so similar; SOX10 is also involved in this control. MET is required for migration of muscle precursor cells from the neural crest into embryonic limb buds, and MyoD and/or Myf5 is a master gene for muscle di erentiation, thus explaining the muscle defects in WS3. People with loss of function mutations in RET often have Hirschsprung disease, but the precise role of the PAX3-RET relationship in WS-HSCR is not clear.

MITF and its mouse homolog mi appear to be master genes for melanocyte development. They encode transcription factors of the basic helix-loop-helix leucine zipper (bHLH-Zip) family. These bind their DNA target as dimers (homoor heterodimers); the basic region a ects the binding and the HLH and Zip regions govern dimerization. Known targets include several melanocyte-specific genes such as tyrosinase and TRP2. Transcription of MITF is under the control of PAX3 and SOX10, and MITF protein is activated by phosphorylation downstream of the KIT receptor (explaining the similarities between WS2 and piebaldism, caused by KIT loss of function mutations) and probably also by GSK3h. MITF protein is also involved in eye development (homozygous mutant mice are microphthalmic) and must have other actions as well, because mice homozygous for certain mi mutations have skeletal and/or mast cell defects.

SOX10 is a member of the large SOX family of transcription factors, the best known member of which is SRY, the male-determining factor. SOX proteins contain an HMG box DNA-binding element, but in vitro this shows weak and very nonspecific binding. Specificity must be achieved by using di erent cofactors that presumably bind to adjacent sequences and then strengthen the DNA binding by protein-protein interaction (45). Exactly how EDNRB/EDN3 mutations cause a WS-HSCR phenotype is not known, though studies of mice carrying manipulable transgenes that could be turned on or o at will have shown that EDN3/EDNRB function is needed only between days 10 and 12.5 to avoid the WS4 phenotype (46).

VII. THE MUTATIONAL SPECTRUM IN

WAARDENBURG SYNDROME

PAX3 attracted interest as the first homeobox gene shown to be mutated in a heritable human disease. Many di erent mutations have been described in WS1 patients, including both truncating (nonsense and splice site) and missense mutations, as well as deletions of the whole gene. This spectrum demonstrates that the pathogenic mechanism is loss of function, and since WS1 is dominant, shows the importance of haploinsu ciency. A completely di erent condition is produced by a gain of function. Alveolar rhabdomyosarcoma, an aggressive childhood tumor, is commonly associated with a translocation t(2;13)(q35;14) that joins PAX3 exons 1–7 on to sequence from the FKHR forkhead-related transcription factor (47). This somatic gene fusion creates a chimeric gene encoding an overactive version of the PAX3 protein (48).

Within WS1 patients there is no significant genotype-phenotype correlation, although careful analysis of a large series (49) showed a higher chance of pigmentary anomaly among patients with premature termination codons upstream of the homeodomain, compared to those with point mutations causing amino acid substitutions in the homeodomain. Unfortunately for genetic counselors, no predictor of hearing loss was found. Nonsense-mediated mRNA decay (50) makes it likely that alleles with premature termination codons produce no PAX3 protein.

Missense mutations are concentrated in sequences encoding two crucial DNA-binding regions, the N-terminal part of the paired domain and the third (recognition) helix of the homeodomain. X-ray crystallography of related proteins has shown that the amino acids involved form crucial DNA contacts (51). Truncating mutations are more widely spread through the gene, as expected. Most are seen only in single families, showing that recurrent mutation is the main mechanism responsible for the persistence of

Waardenburg syndrome. A few recurrent mutations are known. 873insG inserts an extra guanine nucleotide in a run of 6 G’s, and has been seen in several independent families; homopolymeric runs are known to be mutation hotspots because of replication slippage.

Surprisingly, mutations of any sort have almost never been reported from the large region downstream of the homeobox, despite careful analysis of these regions in many patients. This is in contrast to PAX6, where truncating mutations are scattered across the entire coding sequence. The reason for this di erence is hard to discern. This serine-threonine-rich downstream region of the PAX3 protein is presumed to constitute an activation domain. It seems unlikely that mutations in this region should cause no phenotype, or a phenotype so di erent from Waardenburg syndrome that the relevant patients have never been tested.

In a single family the PAX3 missense mutation N47K is associated with a phenotype rather di erent from WS1, craniofacial-deafness-hand syndrome (MIM 122880) (52). It seems likely that amino acid 47 has some special functional significance in the PAX3 protein, because another substitution, N47H, is the cause of the only convincing example of familial WS3 (53).

For MITF, again the spectrum of mutations suggests loss of function and haploinsu ciency as the pathogenic mechanism. Since B-HLH-Zip family proteins act as dimers, there is the possibility of dominant negative e ects, and it seems plausible that such e ects explain two families in which MITF mutations cause Tietz syndrome (54,55). In both cases the basic region is mutated while the HLH and Zip domains remain intact, allowing the mutant protein to form dimers that might well be nonfunctional. The more severe and uniform phenotype of Tietz syndrome contrasts with the great variability of features in WS2-MITF families, the latter being typical of haploinsu ciency. However, we failed to find MITF mutations in several other families with Tietz syndrome (our unpublished data).

SOX10 mutations are rare, but the patients in which they have been found are surprisingly diverse. As well as a few with WS4, there are several with severe neurological features (56) and one described as having a form of the Yemenite deaf-blind-hypopigmentation syndrome (which does not include HSCR) (57). Much remains to be learned about the molecular pathology of SOX10 mutants.

VIII. SUMMARY

The various forms of Waardenburg syndrome give us a window into aspects of neural crest and melanocyte di erentiation. Detailed mechanistic expla-

nations must await better definition of the natural targets of the various transcription factors involved.

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