Charlotte Roze
email: charlotte.roze@linguist.jussieu.fr

Towards a Discourse Relation Algebra for Comparing Discourse Structures

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Introduction

Discourse parsing, the task of producing a discourse structure for a given text, raises two crucial issues: building gold-standard corpora and evaluating discourse annotations. Building a gold-standard generally implies the merging of different annotations of the same text(s) and therefore the comparison of discourse structures produced by different annotators. Evaluating discourse annotations also requires to compare discourse structures generated by a system and those of the reference. But comparing discourse structures is not a trivial task: two discourse annotations of the same text can be different without any of them being wrong or incomplete. For example, the discourse in (1), which contains three elementary discourse units (π 1 ), (π 2 ) and (π 3 ), can receive two different annotations: an annotation A 1 , containing Result(π 1 , π 2 ) and Elaboration(π 2 , π 3 ); an annotation A 2 , containing these two relations plus the relation Result(π 1 , π 3 ). These annotations are equivalent: A 2 can be deduced from A 1 , because Result(π 1 , π 3 ) is implicitly contained in A 1 .

(1) a. It has rained a lot today. (π 1 ) b. So John cooked. (π 2 ) c. He made a pie. (π 3 )

Even though these two annotations are equivalent, if they are compared without deducing the implicit relation, in the case of evaluating a system the score will be different from 1, and in the case where they are to be merged, it's not clear whether the implicit relation must be taken in the gold annotation or not. This example shows that all the implicit information contained in the discourse structures must be deduced in order to improve their comparison. Given a discourse structure associated to a text, we must be able to calculate, using inference rules, the discourse closure of the structure, i.e. all discourse relations that can be inferred from the relations explicitly annotated. To do that, discourse relations inference rules are necessary, based on the semantic constraints that relate different discourse functions. For example, the calculation of the discourse closure of the annotation A 1 requires the following rule:

Result(π 1 , π 2 ) ∧ Elaboration(π 2 , π 3 ) → Result(π 1 , π 3 ).
Despite the interest of using such constraints for a better comparison of discourse structures, interactions between relations and equivalences between structures remain little studied in discourse theories.1 A very small number of discourse relations inference rules have been built. In many cases, we don't know if a typical structure such as R 1 (α, β) ∧ R 2 (β, γ) 2 contains implicit information(s), i.e. implicit relation(s), and if so, which one(s). We therefore propose to study and define discourse relations inference rules, to be integrated into a general framework akin to the algebra of temporal relations put forth by [START_REF] Allen | Maintaining knowledge about temporal intervals[END_REF]. Several theories and formalisms aim at representing discourse structures. In RST (Rhetorical Structure Theory, [START_REF] Mann | Rhetorical structure theory: Towards a functional theory of text organization[END_REF], discourse structures are represented by binary trees [START_REF] Marcu | Building up rhetorical structure trees[END_REF], whose leaf nodes are discourse units and internal nodes are discourse relations. On the other hand, in SDRT (Segmented Discourse Representation Theory, [START_REF] Asher | Logics of Conversation[END_REF], discourse structures are represented by graphs, whose nodes are discourse units and edges are discourse relations. The set of discourse relations also vary according to the different theories and formalisms, as well as the way they are defined. There exists multiplying approaches, such as RST (78 relations in the RST corpus [START_REF] Carlson | Discourse Tagging Manual[END_REF]), which build extended lists of relations, and reductionist approaches, such as the one of [START_REF] Grosz | Attention, intentions, and the structure of discourse[END_REF], which propose to distinguish only two structural relations (dominates and satisfaction-precedence). Nevertheless, discourse relations can be classified into 4 main groups [START_REF] Halliday | Cohesion in English[END_REF]: additive relations, temporal relations, causal relations and adversative relations -corresponding respectively to Expansion, Temporal, Contingency and Comparison in the hierarchy of sense tags from the PDTB (The PDTB [START_REF] Group | The Penn Discourse Treebank 2.0. Annotation Manual[END_REF]. Besides, there is a consensus on a number of relations, such as Narration or Result. Discourse relations can generally be matched from one theory to another: for example, the relation Background from SDRT [START_REF] Asher | Logics of Conversation[END_REF] overlaps the relations Background and Circumstance from RST [START_REF] Mann | Rhetorical structure theory: Towards a functional theory of text organization[END_REF]. We chose to build the inference rules within the framework of SDRT, because this theory makes explicit the semantic constraints established by discourse re-lations on their arguments, and these constraints constitute a starting point for the study of inference rules. Besides, with respect to the definition of discourse relations, this theory is at a level of granularity intermediary between reductionist approaches and multiplying ones. In our work, we focus on discourse relations that can be matched with a relation (or a group of relations) in RST [START_REF] Mann | Rhetorical structure theory: Towards a functional theory of text organization[END_REF][START_REF] Carlson | Discourse Tagging Manual[END_REF] and in the PDTB. For example: Narration (Sequence in RST, Temporal:Asynchronous:precedence in the PDTB), Background (background and circumstance in RST, Temporal:Synchronous in the PDTB), Result (result in the RST corpus, Contingency:Cause:result in the PDTB), etc.

Inference rules have been defined for temporal relations: temporal have been used as inference rules for completing and evaluating temporal graphs [START_REF] Setzer | Using semantic inferences for temporal annotation comparison[END_REF]. For example, if a temporal annotation specifies that the event e 1 happens before e 2 and the event e 2 happens before e 3 , then it implicitly specifies that the event e 1 happens before e 3 . With respect to temporal relations, [START_REF] Allen | Maintaining knowledge about temporal intervals[END_REF] defines a complete temporal algebra, containing rules of the form: r 1 (A, B) ∧ r 2 (B, C) → r 3 (A, C)3 . In many cases, more than one relation r 3 can be deduced between A and C. For example: overlaps(A, B) ∧ overlaps(B, C) → before(A, C)∨overlaps(A, C)∨meets(A, C). Building inference rules for discourse relations is not as easy, since their semantic effects are not as clear as constraints established by temporal relations. To build a discourse relations algebra, two forms of rules (at least) seem necessary. For a discourse with three consecutive discourse units (α), (β) and (γ), there are at least potentially two structures (see figure 1) in which a relation remains undefined (dashed lines in the figure): in the first structure, this is the relation between (α) and (γ) and in the second one, the relation between (β) and (γ). Considering those two structures, we propose two forms of inference rules (see figure 1). 4 The deduction can be a disjunction of relations, as in Allen's temporal algebra. For completion of the algebra, we add an artificial relation None in the set od discourse relations, expressing the fact that two discourse segments are unrelated by a discourse relation, and which is exclusive of other relations.

Building Rules of Inference for Discourse Relations

We detail in this section the methodology we adopt for building discourse relations inference rules. For each premise of a rule, the study is based on the semantic effects of the relations contained in the premise, and the analysis of both intuitively constructed and automatically extracted data. Formulating Candidate Rules At first, we examine the constraints established by the relations contained in the premise of rule under consideration (either R x and R y or R x and R z ), using their theoretical definition, in order to characterize the possible links between the two segments whose relation is unknown (either R z or R y ). The constraints established by the relations of the premise allow us to predict the inference, at least theoretically. For example, the premise Narration(α, β) ∧ Explanation(β, γ) has the following temporal effects: e α < e β and Init(e β ) < Init(e γ ). It leaves several possibilities open about the temporal links between e α and e γ and leads to the formulation of a possible rule: Narration(α, β) ∧ Explanation(β, γ) → Narration(α, γ) ∨ Background(α, γ) ∨ Flashback(α, γ). These three possibilities are illustrated by the discourses in (2), ( 3) and ( 4).

(2) Peter lived in Paris. Then he moved to South because he was offered a post in Marseille. → Narration(α, γ)

(3) Pierre spent two weeks in London, then he went to Greece because it was raining all the time. → Background(α, γ)

(4) Peter went shopping. then he called his sister, because she left him a message yesterday. → Flashback(α, γ)

Studying the constraints established by the relations of the premise also allows for the exclusion of specific relations from possible inferences. For example, for the premise Narration(α, β)∧Narration(β, γ), the inference of Flashback(α, γ) is impossible: given that the eventuality described in (α) happens before the one in (β) and the eventuality described in (β) happens before the one in (γ), the eventuality described in (α) cannot happen after the one in (γ).

The study of inference rules also must get throught an introspective work, i.e. building and analysing discourses containing the premise under consideration, trying to cover the different realizations of the involved discourse relations. This work allows for the verification of the inference hypotheses resulting from the study of semantic effects of discourse relations.

Tests for Identification of Inferences The analysis of discourses containing the premise can be helped by the use of two tests: the insertion of a connective and a discourse reorganization. These tests are used for checking the presence of a specific relation R z between (α) and (γ) in the case of a premise R x (α, β)∧R y (β, γ). The first test is the following: after inserting in the segment (γ) a discourse connective marking R z , if the discourse remains coherent and its interpretation is unchanged, then R z (α, γ) holds. The second test requires the reorganization of the discourse, by inverting the positions of (β) and (γ). The connective marking R y (β, γ) is replaced by a connective marking R y -1 (γ, β) (if the inverse relation R y -1 exists). In the resulting discourse, if a connective marking R z (α, γ) can be inserted, without changing the coherence and the original interpretation of the discourse, then the relation R z (α, γ) holds in the original discourse.

In ( 5) we can see two examples of the insertion test, where we check the presence of the relation Result(α, γ) within the study of the premise Result(α, β)∧ Explanation(β, γ), by inserting the connective therefore: in (5-i) the insertion is possible, and in (5-ii) it is not. The two discourses are reorganized in (6), by inverting the positions of (β) and (γ) and inserting the connective so: the reorganization is possible in (6-i), but it is not in (6-ii).

(5) α. The electricity has been restored this morning.

β. The inhabitants of the building are relieved, γ. (i) because as a result they have been able to return to their homes.

(ii) because (# as a result) they need heating.

(6) α. The electricity has been restored this morning.

γ. (i) So the inhabitants of the building have been able to return to their homes.

(ii) # So the inhabitants of the building need heating.

β. Therefore they are relieved.

Annotation After identifying the possible inferences for a premise, we proceed to a systematic annotation of automatically extracted discourses which contain the premise. For gathering a corpus of discourses containing a specific premise, we developped a tool that identifies the presence of discourse relations by detecting discourse connectives marking them. The extraction is done on the French journalistic corpus Est Républicain and the corpus Europarl, in which syntactic dependencies were annotated beforehand by the Bonsai parser [START_REF] Candito | Analyse syntaxique du français : des constituants aux dépendances[END_REF], using Lexconn, a lexicon of French discourse connectives [START_REF] Roze | Lexconn: a French Lexicon of Discourse Connectives[END_REF] containing 330 connectives collected with their syntactic category and the discourse relation(s) they express. For extracting occurrences of a premise R x (α, β)∧R y (β, γ), the tool detects the following contexts in the corpus:

p 1 p 2 [conn x ] p 3 [conn y ],
where the connective conn x appears in the proposition p 2 and is a marker for R x , and conn y appears in the proposition p 3 and is a marker for R y . In the collected discourses, either conn y is in the same sentence as conn x or in the next sentence. This method only allows for the extraction of discourses where the relations are explicitly marked, but it permits to gather a greater number of examples than in annotated corpora (for example, 2452 discourses were extracted for the premise Explanation(α, β) ∧ Result(β, γ)).

The annotation of discourses contained in the gathered corpus consists in: verifying the presence of the two relations of the premise; if the premise is present in the discourse, using the tests presented in the last section in order to identify an annotate the inferred relation. This annotation allows for the verification of the hypotheses formulated about possible inferences and in the case where a premise gives rise to more than one inference, it is also possible to estimate the frequencies of each result. Within the study of the premise of rule Result(α, β) ∧ Contrast(β, γ), we annotated 171 discourses containing the premise (Table 2). The study of the premise allows the formulation of the rule:

Result(α, β) ∧ Contrast(β, γ) → Contrast(α, γ) ∨ Result(α, γ) ∨ None(α, γ).

Conclusion and Future Work

We proposed a methodology for building discourse relations inference rules, to be integrated into an algebra of these relations. The main objective of building such an algebra is to improve the comparison of discourse structures within the evaluation of discourse annotations and the creation of a gold-standard corpus.

The inference rules can also help detecting inconsistencies in discourse structures, in order to improve human or machine annotation. The premises of rules already studied lead to the formulation of inference rules, established by the theoretical definition of discourse relations, manually constructed data and extracted data. By manually annotating discourses, we also compute inference probabilities. The construction of more inference rules is under way. As for rules that give rise to the deduction of a disjunction of relations, we aim to determine which linguistic features help to predict the inferred relation, based on a linguistic study and an investigation of annotated data by statistical methods. We will also seek to establish generalizations over rules, and attempt to determine whether the type of relation (coordinating or subordinating) has an impact on the inference, and whether relations sharing some semantic effects have similar behavior in inference rules. Another issue is that our study is somewhat limited to discourses where (α), (β) and (γ) are elementary discourse units, so we have to address the following question: are the inference rules still valid when (α), (β) and (γ) are complex discourse units? We are already trying to answer that question within the study of some rules.

Fig. 1 .

 1 Fig. 1. Representation of the two forms of rules: inference of Rz or inference of Ry (where (α), (β) and (γ) are contiguous discourse units)

Table 1 .

 1 The results of the annotation show that in most cases, the relation Contrast is inferred (75%). Percentage

	Inferred Relation Percentage Number
	Contrast	75	128
	Result	15	25
	None	7	12
	Other	6	10
	Total	103	170

of inferred relations between (α) and (γ) in extracted discourses containing the premise Result(α, β) ∧ Contrast(β, γ)

Some inference rules are defined within SDRT (Asher and Lascarides,

2003).For example, there is a transitivity rule for Elaboration: Elaboration(π1 , π2 ) ∧ Elaboration(π2 , π3 ) → Elaboration(π1 , π3 ) (where (π1 ), (π2 ) and (π3 ) are three discourse segments). 2 R1 and R2 represent discourse relations. (α), (β) and (γ) represent discourse units.

A, B et C represent temporal intervals. r1 , r2 and r3 represent temporal relations.

If the relations were temporal ones, the premise Rx (α, β) ∧ Rz (α, γ) would be equivalent to Rx -1 (β, α) ∧ Rz (α, γ), where Rx -1 is the inverse relation of Rx . In Allen's algebra, the premises of rules are all of the form: r1 (A, B) ∧ r2 (B, C). With respect to discourse relations, these two cases must be distinguished because not all discourse relations have an inverse relation, and the ordering of segments in a discourse have consequences on its structure and its interpretation.


Asma Chaoui

Yuli Watanabe

Renaud Touraine

Viviane Baral

Michel Goossens

Véronique Pingault

Nadège Bondurand

Veronique Pingault

Nadege Bondurand
email: nadege.bondurand@inserm.fr

Bondurand Nadège

Identification and functional analysis of SOX10 missense mutations in different

Keywords: Waardenburg syndrome, SOX10, neural crest, enteric nervous system, Hirschsprung's

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Waardenburg syndrome (WS) is an auditory-pigmentary disorder with an incidence of one in 40,000 that manifests with sensorineural deafness and abnormal pigmentation of the hair, skin, and iris [START_REF] Read | Waardenburg syndrome[END_REF]. This syndrome results from an abnormal proliferation, survival, migration or differentiation of neural crest derived melanocytes and is therefore defined as a neurocristopathy [START_REF] Bolande | The neurocristopathies: a unifying concept of disease arising in neural crest maldevelopment[END_REF][START_REF] Douarin | The neural crest[END_REF].

WS is classified into four subtypes, WS1-4, depending on the presence of additional symptoms. The absence of additional features characterizes WS2 (MIM 193510, 611584).

Facial dysmorphic features are present in WS1 and WS3 patients, whereas an association with Hirschsprung's disease (HD, aganglionic megacolon) defines WS4, also called Shah-Waardenburg syndrome or Waardenburg-Hirschsprung's disease (MIM 277580, 613265, 613266). HD is the main cause of congenital intestinal obstruction, with an incidence of one in 5,000 live births. It is characterized by the absence of enteric ganglia along a variable length of the intestine [START_REF] Read | Waardenburg syndrome[END_REF][START_REF] Amiel | Hirschsprung disease, associated syndromes and genetics: a review[END_REF][START_REF] Pingault | Review and update of mutations causing Waardenburg syndrome[END_REF].

WS is a genetically heterogeneous condition. Indeed, numerous heterozygous mutations in the genes encoding the PAX3, MITF, and SOX10 transcription factors, as well as homozygous or heterozygous mutations in the genes encoding endothelin-3 (EDN3) or endothelin-B receptor (EDNRB) have been reported [START_REF] Pingault | Review and update of mutations causing Waardenburg syndrome[END_REF]. Since 1998, about 50 heterozygous mutations or deletions of the SOX10 gene have been described in WS patients [START_REF] Pingault | Review and update of mutations causing Waardenburg syndrome[END_REF]. SOX10 belongs to the SOX (SRY related HMG box) family of transcription factors and is closely related to SOX8 and SOX9, the latter being involved in campomelic dysplasia [START_REF] Foster | Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene[END_REF][START_REF] Wagner | Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9[END_REF][START_REF] Mollaaghababa | The importance of having your SOX on: role of SOX10 in the development of neural crest-derived melanocytes and glia[END_REF][START_REF] Hong | Sox proteins and neural crest development[END_REF][START_REF] Kelsh | Sorting out Sox10 functions in neural crest development[END_REF][START_REF] Guth | Having it both ways: Sox protein function between conservation and innovation[END_REF]. All SOX proteins are characterized by a highly conserved motif of 80 amino acids called the HMG (high mobility group) domain which allows targeting of the DNA minor groove and recognition of a 7 bp consensus DNA element [START_REF] Werner | Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex[END_REF][START_REF] Lefebvre | Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors[END_REF][START_REF] Malki | Shuttling of SOX proteins[END_REF]. The HMG domain is made up of three α-helices that adopt an L-type shape. A hydrophobic core is formed at the intersection of the three helices to maintain the structural shape [START_REF] Murphy | Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation[END_REF][START_REF] Li | SRY-directed DNA bending and human sex reversal: reassessment of a clinical mutation uncovers a global coupling between the HMG box and its tail[END_REF]. The HMG domain also enables intracellular transport regulation [START_REF] Malki | Shuttling of SOX proteins[END_REF]. Indeed, it contains two distinct nuclear localization signals (NLSs) using calmodulin (for the N-terminal NLS) and β importins (for the C-terminal NLS) to drive the SOX proteins into the nucleus [START_REF] Poulat | Nuclear localization of the testis determining gene product SRY[END_REF][START_REF] Sudbeck | Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9[END_REF][START_REF] Malki | Shuttling of SOX proteins[END_REF]. Sex reversal mutations have been independently described in both the N-and C-terminal NLSs of SRY and SOX9; both of these affect the subcellular location of the resulting proteins [START_REF] Sim | Boys, girls and shuttling of SRY and SOX9[END_REF]. Other sequences or post-translational modifications, such as phosphorylation, acetylation, and sumoylation, are also known to modulate the subcellular location of SOX factors [START_REF] Lefebvre | Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors[END_REF][START_REF] Malki | Shuttling of SOX proteins[END_REF]. Besides NLSs, a perfect nuclear export signal (NES) has been identified, suggesting active nucleocytoplasmic shuttling [START_REF] Rehberg | Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation[END_REF].

The distal part of the HMG domain of SOX10 (amino acids 133 to 203) is also involved in protein-protein interactions with its partners and may thus be crucial for synergistic regulation of gene expression [START_REF] Wissmuller | The high-mobility-group domain of Sox proteins interacts with DNA-binding domains of many transcription factors[END_REF]. DNA bindingSOX factors are known to regulate expression of a variety of genes through binding to their promoter or enhancer regions, either alone or in cooperation with various cofactors [START_REF] Kamachi | Pairing SOX off: with partners in the regulation of embryonic development[END_REF][START_REF] Wilson | Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators[END_REF][START_REF] Bernard | Human SRY inhibits beta-cateninmediated transcription[END_REF][START_REF] Kondoh | SOX-partner code for cell specification: Regulatory target selection and underlying molecular mechanisms[END_REF]. SOX10 has thus been shown to play a role in specification and differentiation of the melanocyte lineage by regulating the expression of MITF/Mitf, TRYP2/Dct (Dopachrome tautomerase) and tyrosinase genes, in synergy with PAX3 or MITF [START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF][START_REF] Potterf | Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3[END_REF][START_REF] Jiao | Direct interaction of Sox10 with the promoter of murine Dopachrome Tautomerase (Dct) and synergistic activation of Dct expression with Mitf[END_REF][START_REF] Ludwig | Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf transcription factors[END_REF][START_REF] Murisier | The tyrosinase enhancer is activated by Sox10 and Mitf in mouse melanocytes[END_REF]. It is also crucial for the maintenance of pluripotency of migrating enteric progenitors and their differentiation, which correlates with its regulation of two HD genes: EDNRB and RET, encoding a seven-transmembrane and a tyrosine-kinase receptor respectively [START_REF] Lang | Pax3 is required for enteric ganglia formation and functions with Sox10 to modulate expression of c-ret[END_REF][START_REF] Lang | Sox10 and Pax3 physically interact to mediate activation of a conserved c-RET enhancer[END_REF][START_REF] Zhu | Spatiotemporal regulation of endothelin receptor-B by SOX10 in neural crest-derived enteric neuron precursors[END_REF]Yokoyama et al., 2006b). Finally, SOX10 is essential for the survival and differentiation of glial cells of the peripheral nervous system and for oligodendrocyte development. Its transcriptional targets within these cell lineages include MPZ (myelin protein zero, P0), MBP (myelin basic protein), PLP (proteolipid protein), GJC2 and GJB1 (connexin 47 and connexin 32 respectively; (Peirano et al., 2000;[START_REF] Bondurand | Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10[END_REF][START_REF] Britsch | The transcription factor Sox10 is a key regulator of peripheral glial development[END_REF][START_REF] Stolt | Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10[END_REF][START_REF] Schlierf | Expression of connexin47 in oligodendrocytes is regulated by the Sox10 transcription factor[END_REF]). In the case of connexin 32 (Cx32) activation, SOX10 acts in synergy with the EGR2 transcription factor [START_REF] Bondurand | Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10[END_REF]. Some of these promoters contain multiple SOX10-binding sites, which support either monomeric or cooperative dimeric binding (Peirano et al., 2000;Peirano and Wegner, 2000;[START_REF] Bondurand | Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10[END_REF].

The SOX10 mutations characterized so far are mostly truncating mutations: nonsense or frameshift and two splice mutations, which most often remove all or part of the transactivation domain located in the C-terminal part of the protein, and sometimes the HMG domain [START_REF] Pingault | SOX10 mutations in patients with Waardenburg-Hirschsprung disease[END_REF][START_REF] Pingault | Review and update of mutations causing Waardenburg syndrome[END_REF]. Interestingly, few cases exhibit chronic intestinal pseudo-obstruction instead of HD. In addition to the WS2 and WS4 classic phenotypes, some patients also present with neurological features: peripheral demyelinating neuropathy, central neuropathy, or both, leading to a syndrome called PCWH (Peripheral demyelinating neuropathy-Central dysmyelinating leukodystrophy-Waardenburg syndrome-Hirschsprung's disease) or PCW (PCWH without HD) (MIM 609136 and [START_REF] Inoue | Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations[END_REF][START_REF] Pingault | Review and update of mutations causing Waardenburg syndrome[END_REF]). These more severe phenotypes mostly result from mutations in the last coding exon and have been proposed to occur when the mutant mRNAs escape the nonsensemediated mRNA decay (NMD) pathway [START_REF] Inoue | Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations[END_REF]. However, the recent identification of whole SOX10 gene deletions in PCWH patients can not be explained by this hypothesis [START_REF] Bondurand | Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4[END_REF].

The first SOX10 missense mutation, p.Ser135Thr, was identified in a patient presenting with a peculiar phenotype, the so-called mild form of Yemenite syndrome (reminiscent of WS2 or PCW cases; [START_REF] Bondurand | A molecular analysis of the yemenite deaf-blind hypopigmentation syndrome: SOX10 dysfunction causes different neurocristopathies[END_REF]). In vitro functional studies have suggested that this mutant may differentially influence expression of lineage-specific target genes, accounting for the phenotypic differences observed. Indeed, this mutation abolishes DNA binding but maintains transcriptional activation of RET and EDNRB (and therefore proper enteric development), but not MITF [START_REF] Bondurand | A molecular analysis of the yemenite deaf-blind hypopigmentation syndrome: SOX10 dysfunction causes different neurocristopathies[END_REF][START_REF] Lang | Sox10 and Pax3 physically interact to mediate activation of a conserved c-RET enhancer[END_REF]Yokoyama et al., 2006b;Yokoyama et al., 2006a). Since then, two other missense mutations have been reported, p.Ala157Val and p.Gln174Pro, associated with WS4 and PCW respectively [START_REF] Morin | A de novo missense mutation in the gene encoding the SOX10 transcription factor in a Spanish sporadic case of Waardenburg syndrome type IV[END_REF][START_REF] Barnett | Aplasia of cochlear nerves and olfactory bulbs in association with SOX10 mutation[END_REF]. In the present study, novel SOX10 missense mutations, associated with a variety of phenotypes, ranging from WS2 to WS4 and PCWH, were identified from 11 patients. In addition, the functional effects of all missense mutations described to date and not previously characterized in terms of function were analyzed.

Materials and methods

SOX10 mutation screening:

Informed consent for genetic testing was obtained from patients and/or parents. Genomic DNA was extracted from peripheral blood leukocytes using standard protocols.

Patients A, F, G, and K were screened using dHPLC (primers available upon request). All amplicons showing a difference compared with the controls were then sequenced. The three coding exons of the other patients were analyzed by direct sequencing of the PCR products as previously described [START_REF] Bondurand | Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4[END_REF]. In the absence of a full description of the 5'UTR non-coding exon(s) of SOX10, the exon numbering system previously used was conserved, i.e., the non-coding exons are designated exons 1 and 2, the exon with the ATG codon is designated exon 3, and the exon with the stop codon is designated exon 5. Mutations were named according to international nomenclature based on cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the cDNA reference sequence (GenBank NM_006941.3). To confirm their de novo occurrence, comparison with the parental DNA, along with verification of sample identity, was conducted through analysis of six microsatellites located on six different chromosomes, using the linkage mapping set (Applied Biosystems, Carlsbad, CA, USA).

Bioinformatics and 3D analysis:

The mutations identified were not described as known polymorphisms in the relevant

Plasmid constructs:

The pCMV-SOX10Myc, pECE-SOX10, pECE-E189X, pECE-PAX3, pECE-EGR2, pGL3-MITFdel1718, and pGL3-Cx32 vectors have been previously described [START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF][START_REF] Bondurand | Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10[END_REF][START_REF] Sanchez-Mejias | Involvement of SOX10 in the pathogenesis of Hirschsprung disease: report of a truncating mutation in an isolated patient[END_REF]. The P0 and pSOX10-GFP constructs were kindly provided by M. Wegner (Peirano and Wegner, 2000;[START_REF] Rehberg | Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation[END_REF]. The padRSVkrox20gHA construct was provided by P. Gilardi-Hebenstreit [START_REF] Desmazieres | Krox20 controls the transcription of its various targets in the developing hindbrain according to multiple modes[END_REF]. The Ret enhancer region MSC+9.7wt plasmid was kindly provided by A. McCallion [START_REF] Emison | Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability[END_REF].

All mutations were introduced within the corresponding constructs by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Two different nucleotidic changes are predicted to induce the same p.Met112Ile mutation; of these, the recurrent c.336G>A substitution was chosen for the study. A c.340_341delTGinsGT change was introduced within the pECE-SOX10 vector to create a p.Trp114Val substitution.

Cell culture, transfection, and reporter assays:

HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and transfected using Lipofectamine PLUS reagents (Invitrogen, Carlsbad, CA, USA). Cells were plated on 12-well plates and transfected 1 day later with 0.150 g of each effector and reporter plasmid for the Cx32 promoter analysis or with 0.175 g of each effector and reporter plasmid for the MITF promoter study. The total amount of plasmid was kept constant by the addition of empty pECE vector. Twenty-four hours posttransfection, cells were washed twice with PBS, lysed, and the extracts were assayed for luciferase activity using the Luciferase Assay System (Promega, Madison, WI, USA) as described previously [START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF][START_REF] Bondurand | Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4[END_REF]. In the case of P0, 1.5 g of reporter plasmid was used in combination with 0.400 g of effector, and luciferase activity was measured 72 hours post-transfection. Regarding RET, 0.400 g of reporter and effector plasmids were used, and luciferase activity was assayed 48 hours post-transfection [START_REF] Emison | Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability[END_REF]. For competition assays, increasing amounts of mutant SOX10 plasmids, 0.150 g (1X) or 0.300 g (2X), were mixed with a fixed amount of wild-type SOX10 (0.150 g) and the reporter pGL3-Cx32 plasmid (0.150 g). The DNA per well was kept constant by adding empty pECE vector.

Gel shifts and Western blots:

33 P-labeled S1S2, S5 or C/C' probes (0.5 ng) were incubated with proteins as previously described (Peirano et al., 2000). Experiments were carried out with truncated SOX10 versions (amino acids 1-188) bearing the wild-type or mutated sequences (5 µg / reaction or 10 µg / reaction in case of mutants presenting with nucleocytoplasmic relocalization). Doublestranded oligonucleotides containing one or two SOX10-binding sites from the MITF (S5), Cx32 (S1S2) or P0 (C/C') promoter regions were used as probes (for sequences, see [START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF]Peirano et al., 2000;Peirano and Wegner, 2000;[START_REF] Bondurand | Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10[END_REF]). In some reactions, a specific competitor (C/C'mut probe) was added in a range of 5to 10-fold molar excess. In each case, production of SOX10 proteins was assessed by Western blot using 25 g or 50 g (in case of mutants presenting with nucleocytoplasmic relocalization) of proteins obtained from nuclear extracts (Fig. 2A).

For analysis of protein stability, HeLa cells were transfected with wild-type or mutant SOX10 constructs and treated 24 hours after transfection with 25 g/ml cycloheximide for up to 12 hours. Extracts were prepared after various times of treatment, and SOX10 detected by Western blot. Quantification was performed using the GeneTools v3.05 software.

Immunofluorescence:

HeLa, SKMel5 or N2A cells were plated on 24-well plates and transfected 1 day later with 0.400 µg of expression vectors. Twenty-four hours after transfection, cultures were fixed in 4% paraformaldehyde for 10 minutes at RT and immunohistochemistry was performed as previously described [START_REF] Bondurand | Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4[END_REF] 1 along with the corresponding clinical diagnosis. The two missense variations characterized in patient G, p.Lys150Asn and p.Gly321Arg, occurred de novo. Whether they are on the same or different alleles could not be assessed. Except for p.Gly321Arg, all of these substitutions were located in exon 3 or 4 (see comment in materials and methods regarding exon numbering) and in silico analysis did not predict a major alteration of splicing.

The production of a full-length protein with a single amino acid change remains the most likely outcome in all cases.

As for truncating mutations, most missense mutations occurred de novo. Only two familial cases were observed, with the mutation segregating in several patients over two generations (Table 1). A large variety of phenotypes was observed, ranging from classical WS2 or WS4 to severe PCWH. Of particular interest, the p.Met112Ile substitution was associated with WS2

or PCW/PCWH in independent families. The same c.336G>A change occurred twice independently, a situation rarely observed for SOX10 mutations. Along the same lines, the proline located at position 175 was altered by three distinct changes (patients K, L, and M).

Two other missense mutations were recently published [START_REF] Morin | A de novo missense mutation in the gene encoding the SOX10 transcription factor in a Spanish sporadic case of Waardenburg syndrome type IV[END_REF][START_REF] Barnett | Aplasia of cochlear nerves and olfactory bulbs in association with SOX10 mutation[END_REF], but their functional consequences were not evaluated. These mutations and their associated phenotypes are also presented in Table 1 (patients H and J).

With the exception of p.Gly321Arg, all of these variations were located within one of the main functional domains of the SOX10 protein, i.e., the HMG DNA binding domain (Fig. 1).

ClustalW alignment of the SOX10 HMG box showed that concerned amino acids are fully conserved across evolution (18 species analyzed, data not shown). Moreover, alignment and a variability plot of all human SOX family members showed that these mutations modify relatively conserved amino acids within the HMG box: four out of the nine amino acids concerned are fully conserved among all of the SOX members analyzed, SRY included. An alignment between SOX10 and one member of each SOX subgroup is presented in Fig. 1.

Interestingly, five of the mutations are located in import or export localization signals.

DNA binding and transactivation capacities of the mutant proteins.

To analyze the monomeric and/or dimeric binding capacities of the mutants, electrophoretic mobility shift assays (EMSA) were performed using two different probes: "S5", the SOX10 monomeric binding site from the MITF promoter, and "S1S2", the SOX10 dimeric binding sites from the Cx32 (GJB1) promoter. The previously characterized p.Glu189X mutant was included in all experiments as a positive control. p.Arg106Trp, p.Leu145Pro, p.Lys150Asn, and p.Ala157Val mutants completely lost their DNA binding capacities for both probes (Fig.

2B and C, lanes 3, 6, 7, 8). Other mutants tested retained partial DNA binding capacities for one or both probes, compared with the control (Fig. 2B andC, compare line 2 to lines 5, 9, 10, 11, 12, 13). Interestingly, the p.Met112Ile mutant seemed to present with an increased monomer-binding capacity on the "S1S2" probe (Fig. 2C, lane 4). Quantification of the dimer over monomer ratio confirmed these observations. Indeed, a ratio of 2.4 ± 0.4 and 1.8 ± 0.2 was observed for the p.Glu189X and p.Met112Ile mutants respectively. To confirm this latter observation, binding of this mutant to another well-defined dimeric binding probe, the C/C' probe of the MPZ promoter, was tested. Site C/C' consists of two non-consensus sites with distinct spacing and orientation (Peirano and Wegner, 2000) that favors cooperative binding of two molecules. Its use also suggested that p.Met112Ile presented with increased monomeric binding (Fig. 2D, compare lines 1 and 4). In this case, we observed a dimer over monomer ratio of 5 and 3 respectively. Interestingly, Peirano et al. reported that a mutation of site C' (C'mut probe) resulted in monomeric instead of dimeric binding (Peirano and Wegner, 2000), suggesting that this probe still presented with high affinity to SOX10 monomers.

Therefore, the ability of p.Met112Ile to bind was compared to the control in the presence of the C/C' probe upon C'mut competition. This competition reduced binding of the SOX10 mutant more efficiently than that of the wild-type SOX10 (Fig. 2D, compare lines 4-5 to 2-3), thus confirming increased p.Met112Ile mutant binding as a monomer.

Next, the transactivation potential of each of these mutants was analyzed in vitro. We chose to compare their transactivation capacities on MITF, Cx32, and RET promoters/enhancers, alone or in synergy with their partners, thus testing whether phenotype differences observed among patients could be related to the differential regulation of target genes. We used MITF and Cx32 reporter constructs containing promoter regions of each of these genes. Transactivation depends on SOX10 and PAX3 in case of MITF and SOX10 and EGR2 in case of Cx32 [START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF][START_REF] Bondurand | Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10[END_REF]. As far as RET is concerned, we analyzed the transactivation capacities of the SOX10 mutants on the recently identified SOX10-responsive enhancer located within the RET intron 1 ( [START_REF] Emison | Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability[END_REF] and Fig. 3C).

p.Arg106Trp, p.Leu145Pro, p.Lys150Asn, and p.Ala157Val were unable to transactivate the reporter constructs alone or in synergy with their respective cofactors (Fig. 3A, B, andC).

Other mutants retained partial activity. Indeed, p.Gln174Pro and the 3 p.Pro175 mutants maintained partial or complete activity of the RET enhancer and the MITF promoter, alone or in synergy with PAX3. However, they failed to transactivate the Cx32 promoter, alone or in synergy with EGR2. The p.Arg161His mutant exerted the opposite effects. This mutant was able to activate the Cx32 promoter in a manner similar to that of the wild-type protein, but its activity on MITF and RET was null or much reduced. By contrast, only RET enhancer activity was reduced upon p.Asn131His mutant co-transfection. Finally, the transactivation capacities of the p.Met112Ile mutant were normal for all three reporter genes tested. Based on the results obtained regarding this particular mutant in EMSA experiments using the MPZ probe C/C' (see Fig. 2C), similar studies were performed using the MPZ promoter as a reporter gene. In agreement with its dimeric binding defects, the p.Met112Ile mutant presented with reduced transactivation capability on this promoter (Fig. 3D).

As mentioned above, patient G presented with two missense variations, both of which occurred de novo. The functional relevance of the p.Gly321Arg variation, which is located distal to the HMG domain and outside any known functional domain, was tested alone and in combination with the p.Lys150Asn mutation. p.Gly321Arg maintained full transactivation activity on MITF and Cx32 promoters (Supp.Fig. S1). By contrast, the double mutant presented with null transactivation capacity. These results strongly argue in favor of the p.Lys150Asn mutation as the primary cause of the disease. The p.Gly321Arg variation was therefore not included in further studies.

Previous papers have suggested that the presence or absence of the neurological phenotypes that characterize PCWH syndrome could be related to a nonsense-mediated mRNA decay (NMD) process [START_REF] Inoue | Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations[END_REF]. Most identified mutations lead to truncated proteins with dominant-negative effects in vitro. However, truncating mutations located in the first coding exons (exons 3 and 4) activate the NMD mRNA surveillance pathway and lead to an absence of the mutant protein in vivo (haploinsufficiency), resulting in the classic form of WS4. On the other hand, truncating mutations located in the last coding exon (exon 5) escape nonsense mRNA decay and lead to translation of a mutant SOX10 protein that interferes with the function of the normal SOX10 allele. This dominant-negative effect leads to the more severe PCWH phenotype. The fact that missense mutations are not subject to NMD argues in favor of other mechanisms at the origin of the phenotype variability we observed here. To test the ability of each SOX10 mutant protein to interfere with wild-type SOX10 function, we carried out competition assays by co-transfecting mutant and wild-type SOX10-expressing vectors together with the Cx32 reporter plasmid. As previously described, the p.Glu189X mutant decreased the transcriptional activity of the wild-type protein in a dose-dependent manner (Fig. 3E, [START_REF] Inoue | Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations[END_REF][START_REF] Sanchez-Mejias | Involvement of SOX10 in the pathogenesis of Hirschsprung disease: report of a truncating mutation in an isolated patient[END_REF]). However, none of the missense mutants tested in the present study presented with such an effect (Fig. 3E). According to our experimental results on this specific reporter gene, dominant-negative activity does not appear to correlate with PCWH phenotype observed in several patients.

Subcellular localization of mutant proteins.

We next compared the localization of wild-type and mutant proteins in HeLa cells (Fig. 4), as well as N2A, and SKMel5 cells (data not shown). In all cell types analyzed, wild-type SOX10

protein was distributed in a diffuse pattern throughout the nucleoplasm (Fig. 4, panels A1-A4 and Fig. 5A). p.Met112Ile, p.Asn131His, p.Gln174Pro, and the three p.Pro175 SOX10 mutant proteins retained nuclear localization (Fig. 4, panels C1-4, D1-4, I1-4, J1 to L4). By contrast, the p.Arg106Trp, p.Leu145Pro, p.Lys150Asn, p.Ala157Val, and p.Arg161His proteins were partially redistributed in the cytoplasm (Fig. 4, panels B1-4, E1-4, F1-4, G1-4, H1-4). This redistribution occurred in 80% to 92% of cells transfected cells except for p.Arg161His (37%) (Fig. 5A). Partial cytoplasmic redistribution could therefore be at the origin of the deleterious effects of about half of the mutations identified.

Surprisingly, seven of the mutations induced a striking nuclear redistribution of SOX10 proteins into punctate structures or inclusions that will be called foci within the remaining of the paper. This event, which was not observed upon wild-type construct transfection, occurred irrespective of nuclear or nucleo-cytoplasmic localization. The number of foci ranged from one to greater than 20 per nucleus. Foci observed with the three p.Pro175 mutants were small (from 0.70 to 1.1 m) and detected in 20% of the cells (Fig. 4 J4, K4, L4 and Fig. 5B). Those observed with the p.Leu145Pro, p.Lys150Asn, p.Ala157Val, and p.Gln174Pro mutants were larger (ranging from 1.3 to 6 m) and detected in 26-70% of the transfected cells (Fig. 4 E4, F4, G4, I4, and Fig. 5B). Interestingly, the number and size of foci changed over time. They were observed as early as 3 hours post-transfection and increased in size for up to 48 hours while decreasing in number (data not shown), arguing in favor of foci fusion over time.

Several subnuclear compartments that are enriched in specific proteins have been described in eukaryotic cells [START_REF] Matera | Nuclear bodies: multifaceted subdomains of the interchromatin space[END_REF][START_REF] Matera | Nuclear bodies: random aggregates of sticky proteins or crucibles of macromolecular assembly?[END_REF][START_REF] Carmo-Fonseca | Orphan nuclear bodies[END_REF]. In an attempt to identify the nature of these foci, co-immunolabeling was performed with various markers, such as SC35 for nuclear speckles, PML for PML bodies, and ubiquitin for nuclear aggresomes [START_REF] Fu | Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus[END_REF][START_REF] Dyck | A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptor oncoprotein[END_REF][START_REF] Borden | Pondering the promyelocytic leukemia protein (PML) puzzle: possible functions for PML nuclear bodies[END_REF]. None of these markers co-localized with the SOX10 mutant proteins within the punctate foci. Supp.Fig. S2 shows results obtained for p.Leu145Pro, but similar observations were made for all mutants analyzed. The exact nature of these foci remains to be determined.

Molecular consequences of subnuclear redistribution

Finally, we focused on the molecular and cellular events that could be responsible for foci formation or that could be triggered by foci formation.

Since all SOX10 mutations are dominant, their pathological effects could be due to a gain of function. The competition assay we carried out (Fig. 3E) seems to argue against a dominantnegative effect. Another possibility is that SOX10 missense mutants recruit wild-type SOX10

or interacting partners to the subnuclear foci and thereby exert their deleterious effects. To test this hypothesis, we compared the distribution of a myc-tagged version of wild-type SOX10 upon co-transfection with mutants fused to green fluorescent protein (GFP) or GFP-fused wild-type SOX10. Cells co-transfected with plasmids containing GFP and myc-tagged versions of wild-type SOX10 exhibited uniform nucleoplasmic staining (Supp.Fig. S3, Top panels). Upon wild-type and mutant co-transfection, areas stained by GFP (mutant) and myc (wild-type) were redistributed within foci, indicating that SOX10 mutants could recruit wildtype protein to the punctate foci. Supp.Fig. S3A, bottom panels shows results obtained for p.Leu145Pro, but similar observations were made for p.Ala157Val, p.Gln174Pro, and p.Pro175Arg mutants . On the other hand, co-transfection seemed to decrease the percentage of cells presenting with the punctate pattern, suggesting that wild-type SOX10 expression could partially protect against mutant protein redistribution. In the case of p.Leu145Pro, the percentage of transfected cells containing foci dropped from 34,9 ± 0,3% to 8,1 ± 0,1%.

Similar decreases were observed for all mutants analyzed (data not shown).

We next tested whether expression of SOX10 mutants also resulted in redistribution of cofactors. To this end, we co-transfected wild-type or mutant versions of SOX10 with expression vectors containing PAX3 or EGR2 cDNAs. GFP-tagged SOX10 constructs were used to avoid cross-reactions between antibodies. Diffuse nuclear staining was observed for both cofactors, irrespective of the SOX10 construct co-transfected, suggesting that neither of the two cofactors were redistributed within the foci (Supp.Fig. S3B and data not shown).

Interestingly, the mutations leading to nuclear foci formation were all located within the distal half of the HMG domain (Fig. 6A). This primary structure observation led us to determine the spatial location of the mutant amino acids in 3D reconstructions. As the HMG domain of SOX proteins is highly conserved, we used reconstructions of the closest factors for which structures had been determined by crystallography: SOX17 (67% identity to the SOX10 HMG, 84% similarity, Fig. 6B) and SOX2 (63% identity, 84% similarity) [START_REF] Remenyi | Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers[END_REF][START_REF] Palasingam | The structure of Sox17 bound to DNA reveals a conserved bending topology but selective protein interaction platforms[END_REF]. This led to the interesting observation that several mutations leading to foci accumulation were located either close to or directly surrounding the hydrophobic core that maintains the HMG structural organization (p.Leu145Pro, p.Lys150Asn, p.Ala157Val, Fig. 8B2). By contrast, missense mutations located at the interface with the DNA did not lead to foci formation (p.Arg106Trp, p.Met112Ile, p.Asn131His, Fig. 8B2). The p.Gln174Pro and the three p.Pro175 mutations were in the C-tail of the HMG domain, which is distal to helix 3 and makes contacts both with the N-tail and the DNA minor groove. Their possible functions are more difficult to predict from 3D models (see Fig. 6B3 and Discussion).

These observations led us to propose two hypotheses to explain foci formation: i) an alteration of an unknown functional domain in the distal half of the HMG domain primary structure; or ii) formation of an abnormal tertiary structure resulting in incorrect folding of the protein. To gain insight into these possibilities, we decided to mutate a codon encoding an amino acid that was part of the hydrophobic core but was located within helix 1 of the HMG domain (proximal half of the HMG domain primary structure; p.Trp114Val; see Fig. 6A andB4).

Reminiscent of the effect of mutations surrounding the hydrophobic core, the p.Trp114Val mutant was partially redistributed within large foci (Fig. 6C) and was not able to transactivate MITF or Cx32 promoters, either alone or in synergy with SOX10 cofactors (Fig. 6D). These results indicated that the location of the mutation within the proximal or distal half of the HMG domain was not responsible for foci formation and instead suggested that the foci are induced by alteration of the mutant tertiary structure, possibly resulting in sequestration of malfolded proteins within these foci. To test whether these observations could be related to protein instability, we compared the wild-type and p.Leu145Pro, p.Lys150Asn, p.Ala157Val proteins half life in transfected cycloheximide treated cells and found no significant differences (Fig. 6E).

Discussion

In addition to our first description of a SOX10 missense mutation in the so-called mild form of Yemenite syndrome, we here report 10 new missense mutations in 11 patients presenting with WS2, WS4, or PCW/PCWH ( [START_REF] Bondurand | A molecular analysis of the yemenite deaf-blind hypopigmentation syndrome: SOX10 dysfunction causes different neurocristopathies[END_REF] and the present study). Two missense mutations were also recently published by other groups [START_REF] Morin | A de novo missense mutation in the gene encoding the SOX10 transcription factor in a Spanish sporadic case of Waardenburg syndrome type IV[END_REF][START_REF] Barnett | Aplasia of cochlear nerves and olfactory bulbs in association with SOX10 mutation[END_REF], but their functional relevance was not tested. In this report, the effects of all of these functionally uncharacterized mutations on SOX10 characteristics and functions are described. Table 2 summarizes the results obtained and highlights the deleterious effects of the identified mutations, supporting arguments for their pathogenicity.

With the exception of two mutations, p.Gly321Arg, which is probably not the major cause of the disease in the patient (see Table 1 and supplementary data) and p.Val92Leu (which we previously characterized as non-pathogenic; [START_REF] Bondurand | Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4[END_REF]), all SOX10 missense mutations identified so far are localized in the HMG domain. As a result, some mutants present with partial cytoplasmic and/or subnuclear redistribution, and some lose their DNA binding and transactivation capacities for different tissue-specific target genes. The p.Met112Ile and p.Asn131His mutants were the sole mutants with subtle anomalies. The others presented with one or several gross defects (see Table 2). However, it is sometimes difficult to determine the primary origin of pathogenicity. Several reports have suggested that subcellular localization of SOX factors is finely regulated and essential for their activity [START_REF] Rehberg | Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation[END_REF][START_REF] Lefebvre | Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors[END_REF][START_REF] Malki | Shuttling of SOX proteins[END_REF]. Cytoplasmic redistribution has been linked to SOX-associated pathologies. While some SRY sex-reversing mutations alter both DNA binding and nuclear transport, others affect only nuclear import, suggesting that the latter is the cause of the observed phenotype [START_REF] Argentaro | A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal[END_REF][START_REF] Harley | Defective importin beta recognition and nuclear import of the sexdetermining factor SRY are associated with XY sex-reversing mutations[END_REF][START_REF] Malki | Shuttling of SOX proteins[END_REF]. The partial cytoplasmic redistribution of p.Arg106Trp, p.Leu145Pro, p.Lys150Asn, p.Ala157Val, and p.Arg161His could therefore be considered as the primary defect, despite the null or partial DNA binding and transactivation capacities of these mutants. Interestingly, cytoplasmic redistribution was observed irrespective of the location of the mutations within or outside the NLSs or NES. This was unexpected but may be explained by an indirect effect. Indeed, an abnormal protein configuration induced by a missense mutation distant from any NLS/NES was previously reported in the closely related SOX9 transcription factor, and this mutation resulted in a defective NLS function [START_REF] Preiss | Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation[END_REF][START_REF] Argentaro | A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal[END_REF].

As for SOX10, nonsense/truncating mutations identified in SOX9 and in another SOX family member, SRY, are distributed throughout the coding sequence, while missense mutations are mainly (even if not only) located in the HMG domain (reviewed in [START_REF] Harley | Defective importin beta recognition and nuclear import of the sexdetermining factor SRY are associated with XY sex-reversing mutations[END_REF]). For almost all of the amino acids affected by SOX10 missense mutations, there are equivalent mutated amino acids in SRY and/or SOX9. Of particular interest the p.Pro131Arg mutation of SRY [START_REF] Lundberg | Novel Missense mutation (P131R) in the HMG box of SRY in XY sex reversal[END_REF] and the p.Pro176Leu mutation of SOX9 [START_REF] Michel-Calemard | Campomelic acampomelic dysplasia presenting with increased nuchal translucency in the first trimester[END_REF] are the exact equivalents of the SOX10 mutations p.Pro175Arg and p.Arg175Leu described here. This proline may therefore be a recurrent mutation site in SOX factors. The SOX9 p.Pro176Leu mutation was found in a fetus presenting with sex reversal and acampomelic campomelic dysplasia (ACD) [START_REF] Michel-Calemard | Campomelic acampomelic dysplasia presenting with increased nuchal translucency in the first trimester[END_REF]. In contrast to campomelic dysplasia, ACD was suggested to result from reduced, but not fully abolished, SOX9 function [START_REF] Staffler | Heterozygous SOX9 mutations allowing for residual DNA-binding and transcriptional activation lead to the acampomelic variant of campomelic dysplasia[END_REF]. These observations are consistent with the functional consequences of the SOX10 p.Pro175Leu mutation reported here. A SOX9 mutation p.Met113Val, located on the equivalent of the SOX10 Met112, is also associated with ACD.

This mutant was shown to be located essentially within the nucleus, although a faint cytoplasmic staining was noted. DNA binding and reporter gene activation were reduced [START_REF] Staffler | Heterozygous SOX9 mutations allowing for residual DNA-binding and transcriptional activation lead to the acampomelic variant of campomelic dysplasia[END_REF] and, similar to our observations, modification of the monomeric versus dimeric binding ratio was apparent on the figure. Amino acids affected by missense mutations in various SOX members thus seem to present with similar functional effects. However, their link to specific phenotypes is not clear. In our case, p.Pro175Leu and p.Met112Ile mutations were associated with different phenotypes (PCW vs. WS2, PCWH or PCW, respectively).

As opposed to truncating mutations, which are subject to the NMD pathway, missense mutations located in exons 3 and 4 can lead to PCW/PCWH. Searching for other mechanisms responsible for phenotype variability was therefore of prime interest. The p.Ser135Thr mutation was the only missense mutation described so far in terms of functional effects.

Several studies have suggested that this mutant may differentially influence tissue-specific gene expression, accounting for the phenotypic differences observed [START_REF] Lang | Sox10 and Pax3 physically interact to mediate activation of a conserved c-RET enhancer[END_REF]Yokoyama et al., 2006b;Yokoyama et al., 2006a). In an attempt to identify molecular mechanisms underlying the phenotype variability observed in our patients, we performed similar experiments by testing the transactivation capacities of mutant proteins on melanocytic (MITF), enteric (RET) and glial (Cx32) gene expression. No correlation between tissue-specific promoter activation and the phenotypes observed was evident. Indeed, despite the fact that six of the mutants studied maintained complete or partial activity of the MITF promoter, all patients presented with pigmentation defects. Along the same lines, the p.Met112Ile mutation did not alter the capacity of SOX10 to activate the Cx32 promoter; however, two out of the three patients harboring this mutation presented with severe neurological defects (see Tables 1 and2). No correlation between RET activation and ENS defects was apparent either. The differential tissue-specific target gene transactivation observed in vitro does not seem to be the main basis for phenotype variability observed here.

We finally considered the possibility that SOX10 mutations associated with severe phenotypes could exert dominant-negative effects on wild-type SOX10. Within the limits of our experimental assay, competition assays did not favor this hypothesis. The molecular origin of phenotype variability thus remains to be established.

One of the most striking findings of our study was the observation that seven of the mutants analyzed presented with altered subnuclear distribution within structures that we called foci.

The nuclei of eukaryotes contain various structures that have been characterized

morphologically and are collectively referred to as "nuclear bodies". The wide variety of components that are concentrated within these structures makes them a likely interface for multiple cellular processes, including transcription, RNA processing, transport, protein modification, apoptosis, and cell cycle control [START_REF] Matera | Nuclear bodies: multifaceted subdomains of the interchromatin space[END_REF][START_REF] Matera | Nuclear bodies: random aggregates of sticky proteins or crucibles of macromolecular assembly?[END_REF]. SOX factors were previously found to be associated with some of these structures upon posttranslational modifications such as phosphorylation, acetylation and sumoylation [START_REF] Sachdev | PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies[END_REF][START_REF] Thevenet | Regulation of human SRY subcellular distribution by its acetylation/deacetylation[END_REF][START_REF] Fernandez-Lloris | Repression of SOX6 transcriptional activity by SUMO modification[END_REF][START_REF] Lefebvre | Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors[END_REF]. In context of the latter, it was previously shown that SOX6 and Sumo 2 co-expression results in the appearance of SOX6 in a punctate pattern that co-localizes with PML but which is partially abolished by alteration of SOX6 sumoylation sites [START_REF] Fernandez-Lloris | Repression of SOX6 transcriptional activity by SUMO modification[END_REF]. In our case, preliminary results indicated that co-transfection with Sumo 1, 2, or 3, or alteration of SOX10 sumoylation sites did not appear to modify the punctate relocation. SRY and SOX6

have also been associated with splicing factors such as SC35 [START_REF] Ohe | A direct role of SRY and SOX proteins in pre-mRNA splicing[END_REF]. In our study, none of the SOX10 mutants co-localized with this marker, suggesting that the foci observed were not nuclear speckles. However, previous results suggested that SOX9 interacts with another nuclear RNA binding protein (p54 nrb ) within paraspeckels, an interaction that links SOX9-dependent transcription to target gene mRNA maturation during chondrogenesis [START_REF] Hata | Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice[END_REF]. Whether similar events could explain our observations should be tested in the near future.

Whether these foci are the main cause of pathogenic effects or a consequence of them is not clear, but such subnuclear relocalization has not been extensively described in functional studies of other SOX gene mutations identified in patients. To our knowledge, only the polyalanine expansion mutations identified in SOX3 were reported to generate cellular aggregates [START_REF] Wong | Polyalanine expansion mutations in the X-linked hypopituitarism gene SOX3 result in aggresome formation and impaired transactivation[END_REF]. The type of mutations we identified is very different and our results argue against similar mechanisms at the origin of accumulations. Outside of the SOX family, mutations in other transcription factors have been shown to modify subnuclear localization. An R288P mutation within the DNA binding domain of Maf, which had been shown to cause cataracts, eliminated the transcriptional activity of this transcription factor without altering DNA binding. This mutant was enriched in nuclear foci of unknown origin [START_REF] Rajaram | Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract[END_REF] and recruited SOX as well as other interaction partners within these structures, providing a potential explanation for the dominant disease phenotype. In our case, foci observations were not directly linked to loss of DNA binding or transactivation capacities. We also determined whether cofactors could be recruited within nuclear foci, but none of the results argued in favor of this hypothesis. Based on 3D modeling, we finally considered the possibility that some of these "punctate" mutants, namely p.Leu145Pro, p.Lys150Asn, and p.Ala157Val, disrupt the tertiary structure of the HMG domain.

Interestingly, a similar situation has been described for homeodomain transcription factors.

The homeodomain is composed of three α-helices folded around a hydrophobic core that maintains the 3D conformation. Missense mutations have been mainly localized to DNA contact sites and within the hydrophobic core, resulting in either altered DNA binding or destructuration as the main cause of the disease phenotypes [START_REF] Chi | Homeodomain revisited: a lesson from disease-causing mutations[END_REF]. In our case, all three mutations may affect formation of the hydrophobic core without obviously affecting protein stability, resulting in sequestration of malfolded proteins within foci.

Interpretation is more difficult with the p.Gln174 and p.Arg175 mutations. Gln174 is specific to the SoxE subgroup proteins and thus is not found in the SOX2 and SOX17 HMG domains we used as models. In addition, these two amino acids are located close to the terminal end of the domain used for crystallography. They may not, therefore, be fully representative of the structure in the whole SOX proteins. This HMG C-tail, the structure of which is one of the most variable among HMG domains, has been shown to change conformation upon DNA binding [START_REF] Palasingam | The structure of Sox17 bound to DNA reveals a conserved bending topology but selective protein interaction platforms[END_REF] to make a secondary hydrophobic core and establish several contacts with the minor groove [START_REF] Murphy | Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation[END_REF]. As a result, these mutations may affect DNA binding or 3D structure. Other hypotheses could also explain their pathogenicity. They all create or remove a proline in the C-tail, possibly leading to an abnormal position of the whole carboxy end of the protein. They could also affect binding to cofactors as the distal part of the HMG domain is involved in contacts with cofactors [START_REF] Wissmuller | The high-mobility-group domain of Sox proteins interacts with DNA-binding domains of many transcription factors[END_REF].

However, our experimental results do not favor this hypothesis, as two of the p.175 mutants retained synergistic activity with PAX3.

Very few SOX10 missense mutations have been characterized so far. In this study, we show that such mutations are not as rare as previously described. As opposed to the hypotheses made in the context of the p.Ser135Thr mutant study [START_REF] Bondurand | A molecular analysis of the yemenite deaf-blind hypopigmentation syndrome: SOX10 dysfunction causes different neurocristopathies[END_REF][START_REF] Lang | Sox10 and Pax3 physically interact to mediate activation of a conserved c-RET enhancer[END_REF], in the present study, we found no argument for a link between the activation of tissue-specific promoters/enhancers in vitro and the resulting phenotype. However, the observation that the p.Met112Ile mutation is recurrent (a very rare situation in SOX10) and leads to different phenotypes opens the possibility that the genetic background is influential, as is often suggested for neurocristopathies, and Hirschsprung's disease in particular [START_REF] Gabriel | Segregation at three loci explains familial and population risk in Hirschsprung disease[END_REF][START_REF] Chakravarti | Hirschsprung disease. The metabolic and molecular bases of inherited disease[END_REF][START_REF] Mccallion | Genomic variation in multigenic traits: Hirschsprung disease[END_REF][START_REF] Amiel | Hirschsprung disease, associated syndromes and genetics: a review[END_REF]. Finally, the redistribution of several missense SOX mutant proteins into nuclear foci is intriguing. The characterization of their molecular nature may help to clarify whether they are a cause or a consequence of mutation-related pathogenicity, and furthermore, will provide opportunities for enhanced understanding of the function and mode of action of SOX10 and possibly other SOX factors.

Figure legends Figure 1 .

 1 Figure legends Figure 1. Location of SOX10 missense mutations identified. Schematic representation of

Figure 2 .

 2 Figure 2. DNA binding capacities of the mutant proteins. (A) Western blots presenting

Figure 3 .

 3 Figure 3. Transactivation capacities of the mutant proteins. MITF (A) or Cx32 (B)

Figure 4 .

 4 Figure 4. Subcellular localization of the mutant proteins. HeLa cells transfected with wild-

Figure 5 .

 5 Figure 5. Quantification of the effect of the mutations on the cellular distribution of

Figure 6 .

 6 Figure 6. Origin of foci formation. (A) Location of the mutations leading to nuclear foci

Figure1Figure4Figure5Figure6

  Figure1

Table 1 : Summary of missense mutations identified and clinical findings. Mutations indicated in bold are new identified mutations. Non bold mutations were previously published (see references). cDNA numbering follows the reference NM_006941.3. Two familial cases are reported (D and L). Gender, age and phenotype of each family member are reported. HD=

 1 Hirschsprung; WS=Waardenburg syndrome; po= intestinal pseudo obstruction. M: Months; ND=Not Determined.

	Case Nucleotide change	Protein (predicted)	Inheritance Gender Age	Phenotype	depigmentation hearing loss	Intestinal	Neurological	References
	A	c.316C>T	p.Arg106Trp	ND	F	20	WS4	+	HD	-	This paper
	B	c.336G>A	p.Met112Ile	de novo	M	1	PCWH	+	HD	+	This paper
	C	c.336G>A	p.Met112Ile	de novo	M	7 M	PCW or PCWH	+ (hypoacousia one side)	temporary constipation	+	This paper
	D	c.336G>C	p.Met112Ile	familial	F/F	43/13	WS2 (2/2)	+ (2/2)	-	-	This paper
	E	c.391A>C	p.Asn131His	de novo	M	2	PCWH	+	HD	+	This paper
	F	c.434T>C	p.Leu145Pro	? (Father not tested)	M	3.5	WS4	+	HD	-	This paper
	G	c.[450G>C(+)961G>C] p.[Lys150Asn(+)Gly321Arg] de novo	M	4	PCWH	+	HD	+	This paper
	H	c.470C>T	p.Ala157Val	de novo	M	18 M	WS4	+	HD	-	(Morin et al., 2008)
	I	c.482G>A	p.Arg161His	ND	M	2	WS2	+	-	-	This paper
	J	c.521A>C	p.Gln174Pro	?	M	18 M	PCW	+	-	+	(Barnett et al., 2009)
	K	c.523C>G	p.Pro175Ala	de novo	F	11	PCWH	+	po	+	This paper
	L	c.524C>T	p.Pro175Leu	familial	F/3M 50/44/11/10 PCW (4/4)	+ (4/4)	-	+ (4/4)	This paper
	M	c.524C>G	p.Pro175Arg	de novo	M	30	PCWH	+	constipation +	This paper

Acknowledgments:

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and Agence Nationale de la Recherche (ANR-JCJC).

We would like to thank Natacha Martin and Bruno Costes for sequencing, Xavier Decrouy for confocal microscopy. Drs JL. Alessandri (Saint-Denis, La Reunion, France), J. Amiel (Paris, France), A. Colley (Liverpool, Australia), A. Fryer (Liverpool, UK), B. Gilbert (Poitiers, France), M. Holder (Lille, France), A. Jacquette (Paris, France), S. Manouvrier (Lille, France), L. Pierre-Louis (Fort-de-France, France), P. Uldall (Copenhagen, Denmark) for providing patient data. of SOX10-GFP expression vector co-transfected with the expression vector encoding EGR2-HA or PAX3 cofactors to compare their intracellular distribution. Cells were counterstained with TO-PRO-3-iodide to visualize nuclei (blue). White arrows indicate nuclear foci. Antibodies used are as follow: anti-HA (C12CA5; Roche Applied Science, Indianapolis, IN, USA) 1:75; The PAX3 and myc (9E10) monoclonal antibodies developed by Charles P. Ordahl and J. Michael Bishop were obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242, and used at 1:200 and 1:50 dilutions, respectively.