François-Régis Chaumartin
email: fchaumartin@linguist.jussieu.fr

UPAR7: A knowledge-based system for headline sentiment tagging

For the Affective Text task at SemEval-2007, University Paris 7's system first evaluates emotion and valence on all words of a news headline (using enriched versions of SentiWordNet and a subset of WordNet-Affect). We use a parser to find the head word, considering that it has a major importance. We also detect contrasts (between positive and negative words) that shift valence. Our knowledge-based system achieves high accuracy on emotion and valence annotation. These results show that working with linguistic techniques and a broad-coverage lexicon is a viable approach to sentiment analysis of headlines.

Introduction

Objectives

The detection of emotional connotations in texts is a recent task in computational linguistics. Its economic stakes are promising; for example, a company could detect, by analyzing the blogosphere, people's opinion on its products.

The goal of the SemEval task is to annotate news headlines for emotions (using a predefined list: anger, disgust, fear, joy, sadness & surprise), and for valence (positive or negative). A specific difficulty here is related to the small number of words available for the analysis.

Overall architecture

Our system is mainly rule-based and uses a linguistic approach. From a macroscopic point of view, we follow the hypothesis that, in a news title, all the words potentially carry emotions. If linguistic resources make it possible to detect these emotions individually, how can we deal with headlines where bad and good emotions appear at once?

Our objective is to identify the expression which carries the main topic of the title. One can consider that this expression has a primary importance.

We also seek to lay down rules for detecting specific emotions. For instance, surprise sometimes comes from the contrast between good and bad news. And sometimes, simple lexical elements are characteristic of an emotion; a negation or a modal auxiliary in a title may be a relevant indicator of surprise.

We describe here the techniques we implemented to address all these points.

Components & resources used

The system we employed for the Affective Text evaluation consists of the following components1 :

• The SS-Tagger (a Part-of-Speech tagger)2 , •

The Stanford Parser.

We also used several lexical resources:

• WordNet version 2.1, • A subset of WordNet-Affect, • SentiWordNet.
As the SS-Tagger is straightforward, we will not say more about it here. We will, however, discuss the remaining components and resources below.

Choice of the Stanford Parser

We wished to use a syntactic parser for this task. We hesitated between two parsers producing a dependency graph, the Link Grammar Parser [START_REF] Sleator | Parsing English with a Link Grammar[END_REF] and the Stanford Parser [START_REF] Manning | Fast Exact Inference with a Factored Model for Natural Language Parsing[END_REF].

As a news title is sometimes reduced to a nominal group, without a verb, our experiments showed that we should modify the title to make it "grammatically correct". Such a step is essential to obtain accurate results with a rule-based analyzer such as the Link Grammar Parser. On the other hand, a statistical analyzer like the Stanford Parser is more tolerant with constructions which are not grammatically correct. That is why we chose it.

WordNet

We used WordNet [START_REF] Miller | WordNet: A lexical database[END_REF] as a semantic lexicon. This well-known project, started in 1985 at Princeton, offers a broad-coverage semantic network of the English language, and is probably one of the most popular NLP resources.

In WordNet, words are grouped into sets of synonyms. Various semantic relations exist between these synsets (for example, hypernymy and hyponymy, antonymy, derivation…).

WordNet-Affect

WordNet-Affect [START_REF] Strapparava | Word-Net-Affect: an affective extension of WordNet[END_REF]) is a hierarchy of "affective domain labels", with which the synsets representing affective concepts are further annotated. We used the subset of WordNet-Affect provided as emotions lists by the SemEval organizers. To improve it, we manually added to the emotion lists new words that we found important on the task trial data.

Nouns The synsets of emotions lists were considered as seeds; our system recursively propagated their emotions to their neighbor synsets 3 .

SentiWordNet

SentiWordNet [START_REF] Esuli | SentiWordNet: A Publicly Available Lexical Resource for Opinion Mining[END_REF] describes itself as a lexical resource for opinion mining. SentiWordNet assigns to each synset of WordNet three sentiment scores 4 : positivity, negativity, objectivity, the sum of which always equals 1.0.

This resource has been created with a mix of linguistics and statistics (using classifiers). The advantage of this approach is to allow the automatic generation of emotion values for all the synsets of WordNet. The disadvantage is that, as all the results are not manually validated, some resulting classifications can appear incorrect 5 .

We recursively propagate the positivity and negativity values throughout neighbor synsets 6 .

UPAR7 Affective Text system

"De-capitalization" of common words

A preliminary problem that we had to solve was related to the Anglo-Saxon habit of putting initial capital letters in all the words of a title.

The first pass of our system thus detected news titles that were "improperly" capitalized, and "decapitalizes" their common words.

For that, we used the SS-Tagger on the title; according to the part of speech of each word, information found in WordNet, and some hand-crafted rules 7 , the system chooses or not to keep the initial.

The impact of this processing step is far from negligible, from the point of view of the Stanford Parser. Indeed, let us have a look at the difference between the parsing of the title, before (figure 1) and after (figure 2) this processing.

3 Following relations such as Hyponym, Derivation, Adjective Similar, Adjective Participle, Derivation and Pertainym. 4 For instance, the synset ESTIMABLE#1 (deserving of respect or high regard) has: Positivity = 0.75, Negativity = 0.00, Objectivity = 0.25. 5 For example, RAPE#3 (the crime of forcing a woman to submit to sexual intercourse against her will) is classified with Positivity=0.25 and Negativity=0.0 despite the presence of the word "crime" in its gloss. 6 Using WordNet's relations such as Hyponym (for noun and verb), Antonym and Derivation. For antonyms, positivity and negativity values are exchanged. 7 For instance, a word that cannot be any form of a WordNet lemma is probably a proper noun, and then we keep its initial.

Individual words rating

For the moment, we consider the output of the Stanford Parser as an array of PoS-tagged words. We use WordNet's morphology functions to find the possible base form of each word.

At this stage, an important question arose: was lexical disambiguation possible? We thought not, because with short sentences, few relevant heuristics apply. We chose another solution, by considering that the emotion and valence values of a word were the linear combination of that of all its possible meanings, balanced by the frequency of each lemma.

We detected emotion and valence values for each word, by using our enriched version of WordNet-Affect and SentiWordNet.

In fact, we also detected some extra information:

• An additional 7 th emotion, that looks like "compassion for people needing protection".

Our assumption is that certain words express a subjacent need for protection. For example, there is "student" behind "school", and "child" behind "adoption". So, we built a list of words designating something that needs protection; we also include in this list words such as "troops", "shoppers"…

•

We tried to detect acronyms relating to technology; for this, we defined a list of high-tech companies and a very basic regular expression rule saying that a word (not in WordNet) containing numbers, or capitals not in first position, should be something high-tech. (This very basic rule seems to work nicely on PS3, iPod, NASA…). We use these high-tech indications to increase the "joy" emotion.

•

We counted lexical elements that we think are good indicators of surprise: negations, modal auxiliaries, question marks.

At this stage, we begin some post-processing on individual words. Which factors cause anger rather that sadness? We believe that human intention (to harm) causes the former emotion, while natural factors such as disease or climatic catastrophes cause the latter. So, we used a few rules related to the WordNet noun hierarchy, based on the fact that when a noun is a hyponym of a given synset, we boost some emotions:

Global sentence rating

At this stage, our system tries to find the main subject of the news title. Again, we use the output of the Stanford Parser, but this time, we make use of the dependency graph. We consider that the main word is the root of the dependency graph, i.e. the word that is never a dependant word. (For instance, in figure 2, the main word is "predicts".)

We think that the contribution of this main word is much more important than that of the other words of the title8 . So, we multiply its individual valence and emotion by 6.

The last important part of linguistic processing is the detection of contrasts and accentuations between "good" or "bad" things. We search patterns like [noun subject verb] or [verb direct object noun] in the dependency graph, with verbs that increase or decrease a quantity9 . Using the valence of the given noun, this gives our system the ability to detect very good news ("boosts (brain) power") or good news where something bad gets less important ("reduces risk", "slows decline", "hurricane weakens"…). The valence detection accuracy (55% in coarsegrained evaluation) is lower than in emotion annotation. We attribute this difference to the fact that it is easier to detect emotions (that are given by individual words) rather than valence, which needs a global understanding of the sentence.

Results

Coase

Conclusion

Emotion and valence tagging is a complex and interesting task. For our first attempt, we designed and developed a linguistic rule-based system, using WordNet, SentiWordNet and WordNet-Affect lexical resources, that delivers high accuracy results. In our future work, we will explore the potential of simultaneously using a statistical approach, in order to improve recall of sentiment annotation.

Figure 1 :

 1 Figure 1 : Output of the Stanford Parser with a title that is "improperly" capitalized.

Figure 2 :

 2 Figure 2 : Output of the Stanford Parser with a title that is "properly" capitalized. (Words are tagged with the right part-of-speech, and dependencies are now correct.)

Table 1 :

 1 Counting of new words for each emotion.

		Verbs Adjectives Adverbs
	Anger	37	26	16	0
	Disgust	35	19	9	0
	Fear	71	26	20	4
	Joy	50	22	14	1
	Sadness	88	37	29	4
	Surprise	16	29	13	2
	Nouns	Verbs	Adjectives Adverbs
	cancer	demolish	comatose	bloody
	danger	injure	nuclear	dead
	poverty	kidnap	violent	worse

Table 2 :

 2 Some words added for "fear" emotion.

Table 4 :

 4 Emotions that change valence.

	Does noun inherit from?	Emotions to boost
	UNHEALTHINESS	Fear, sadness
	ATMOSPHERIC PHENOME-	Fear, sadness
	NON
	AGGRESSION, HOSTILITY,	Anger, fear, sadness,
	WRONGFUL CONDUCT	disgust
	WEAPONRY, WEAPON	Anger, fear, sadness
	SYSTEM
	UNFORTUNATE PERSON	Sadness, "compassion"
	HUMAN WILL	Anger
	Table 3: Hypernyms triggering an emotion boost.
	Then, the emotions found serve to update the
	valence, by increasing positivity or negativity:
	Emotion	Positivity Negativity
	Joy	++	--
	Anger, disgust, sadness,	--	++
	fear, "compassion"

Table 5 :

 5 Results of the emotion annotation.

	Fine-grained		-grained
	Pearson Accuracy Precision Recall
	Anger	32.33	93.60	16.67	1.66
	Disgust	12.85	95.30	0.00	0.00
	Fear	44.92	87.90	33.33	2.54
	Joy	22.49	82.20	54.54	6.66
	Sadness	40.98	89.00	48.97 22.02
	Surprise	16.71	88.60	12.12	1.25
	Our rule-based system detects the six emotions
	in news headlines with an average accuracy reach-
	ing 89.43% (coarse-grained evaluation). However,
	recall is low.
	Fine-grained	Coase-grained
	Pearson Accuracy Precision Recall
	Valence	36.96	55.00	57.54	8.78

Table 6 :

 6 Results of the valence annotation.

We used them through the Antelope NLP framework (www.proxem.com), which makes them easy to use.

This fast PoS tagger uses an extension of Maximum Entropy Markov Models. See[START_REF] Tsuruoka | Bidirectional Inference with the Easiest-First Strategy for Tagging Sequence Data[END_REF].

In sentences like "study says…", "scientists say…", "police affirm…", the main head word is the verb of the relative.

We "rediscovered" valence shifters (words that modify the sentiment expressed by a sentiment-bearing word, see[START_REF] Polanyi | Contextual Valence Shifters[END_REF]).


Dr Claire Julian-Reynier
email: claire.julian-reynier@inserm.fr

Julien Mancini

Emmanuelle Mouret-Fourme

Marion Gauthier-Villars

Valérie Bonadona

Pascaline Berthet

Jean-Pierre Fricker

Olivier Caron

Elisabeth Luporsi

Catherine Nogues

Marion Gauthier- Villars

Catherine Noguès

Cancer risk management strategies and perceptions of unaffected women five years after predictive genetic testing for BRCA1/2 mutations

Keywords: Risk perception, BRCA1/2 genetic testing, preventive management, Breast/ovarian cancer

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

Many surveys have addressed the short-term impact of BRCA1/2 genetic mutation testing on perceived risks and behavioural outcomes in heterogeneous samples [START_REF] Scheuner | Delivery of genomic medicine for common chronic adult diseases: a systematic review[END_REF][START_REF] Heshka | A systematic review of perceived risks, psychological and behavioral impacts of genetic testing[END_REF] . In three prospective studies, unaffected women were followed for two [START_REF] Meijers-Heijboer | Presymptomatic DNA testing and prophylactic surgery in families with a BRCA1 or BRCA2 mutation[END_REF][START_REF] Botkin | Genetic testing for a BRCA1 mutation: prophylactic surgery and screening behavior in women 2 years post testing[END_REF] and 3 years 5 after disclosure of their test results in a routine context, and only one study has followed a selected sample of 23 women (21 of whom underwent risk reducing mastectomy) up to 5 years [START_REF] Van Oostrom | Long-term psychological impact of carrying a BRCA1/2 mutation and prophylactic surgery: a 5-year follow-up study[END_REF] . The largest samples of unaffected BRCA1/2 carriers studied so far included 53 [START_REF] Foster | Predictive genetic testing for BRCA1/2 in a UK clinical cohort: three-year follow-up[END_REF] and 68 women [START_REF] Meijers-Heijboer | Presymptomatic DNA testing and prophylactic surgery in families with a BRCA1 or BRCA2 mutation[END_REF] . In these studies, the behaviour of cohorts of female non-carriers from BRCA1/2 families was followed simultaneously. Another study was specifically designed to describe the behaviour of this group [START_REF] Dawson | Cancer risk management practices of noncarriers within BRCA1/2 mutation positive families in the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer[END_REF] . A large Canadian survey retrospectively selected a large number of unaffected BRCA1/2 carriers (N=342) and followed their preventive behaviours until 4 years after their test results on average [START_REF] Metcalfe | Variation in rates of uptake of preventive options by Canadian women carrying the BRCA1 or BRCA2 genetic mutation[END_REF] .

A decrease in breast and ovarian cancer risk perception has been described in non-carriers 12 months after genetic testing, and a lower breast cancer risk perception in non-carriers compared to carriers. Only one study has shown that carriers' risk perceptions were higher six months after disclosure than before testing 9. . It has been concluded that risk perception decreases with time 2 . Behavioural outcomes have been described as rates of surveillance or prophylactic surgery undergone by carriers/non-carriers: Risk Reducing Mastectomy (RRM) is undergone by most carriers in the Netherlands (55%) [START_REF] Meijers-Heijboer | Presymptomatic DNA testing and prophylactic surgery in families with a BRCA1 or BRCA2 mutation[END_REF] and Denmak [START_REF] Skytte | Risk-reducing mastectomy and salpingo-oophorectomy in unaffected BRCA mutation carriers: uptake and timing[END_REF] , in a third of the UK series studied (34%) [START_REF] Foster | Predictive genetic testing for BRCA1/2 in a UK clinical cohort: three-year follow-up[END_REF] and less frequently elsewhere [START_REF] Botkin | Genetic testing for a BRCA1 mutation: prophylactic surgery and screening behavior in women 2 years post testing[END_REF][START_REF] Metcalfe | Variation in rates of uptake of preventive options by Canadian women carrying the BRCA1 or BRCA2 genetic mutation[END_REF][START_REF] Lynch | Patient responses to the disclosure of BRCA mutation tests in hereditary breast-ovarian cancer families[END_REF] . Although the rates of Risk Reducing Salpingo-Oophorectomy (RRSO) reported were higher, most of these studies made no reference to the age at which the intervention should be proposed according to current guidelines. Rates of compliance with mammographic surveillance have been studied in women who were screened for two years after test disclosure, but no information is available about the number of investigations carried out. Women's decision-making processes have been described individually but no information has been published so far about the follow-up of various age-groups or about the whole series of decisions on which their medical management depends. In particular, the uptake of Magnetic Resonance Imaging (MRI) by these cohorts as a means of surveillance has not yet been documented.

The aim of this study was first to describe the sequences of preventive decisions made by women up to five years after disclosure of their test results and the surveillance/surgical options chosen by various age-groups. It was also proposed to examine the impact of BRCA1/2 genetic testing and preventive strategies on these women's cancer risk perceptions with time. Women included in this prospective national study were unaffected carriers/non-carriers who underwent a baseline assessment before test result disclosure and regular follow-up for the next 5 years. BRCA1/2 carriers' and non-carriers' long-term risk perceptions were expected to depend not only on their carrier/non-carrier status but also on the series of preventive strategies chosen.

M Ma at te er ri ia al l a an nd d M Me et th ho od ds s

Population

In the framework of the ongoing French "Genepso" project managed by the French Cancer Genetic Network, BRCA1/2 carriers attending 29 participating centres were registered between 2000 and 2006. Only epidemiological data have been published so far on this cohort, although other studies are under way 12,13 14 . In the present study on "Psychosocial and Preventive Behaviour", non-carriers from families where these BRCA1/2 mutations had been identified were included along with carriers.

The behavioural characteristics of unaffected female carriers and non-carriers of the deleterious BRCA1/2 mutation were therefore compared. For this purpose, all the cases who reached the five year follow-up endpoint were selected. In France, the cost of cancer genetic consultations, BRCA1/2 testing and any subsequent medical management of people at risk is covered by the national healthcare system.

Questionnaires

Process

Consultees were asked to complete six self-administered questionnaires. The first questionnaire, which was designed to collect baseline information, was completed before disclosure of the BRCA1/2 genetic test results by all the women. The women who agreed to participate to the follow-up were then given a second questionnaire at disclosure, to be returned fifteen days later. The 4 other questionnaires were mailed to the women's homes 6 months, one year, 2 years and 5 years later. If no answer had been received one month after mailing a questionnaire, a reminder letter and a copy of the questionnaire were sent out. All the questionnaires were mailed back to the coordinating centre. The procedure used was approved by the "Comité National Informatique et Libertés".

The cancer geneticists also completed a questionnaire describing the women's family and medical characteristics at inclusion and follow-up.

Variables

Preventive behavior

At the five-year follow-up, information was collected about the occurrence, frequency and number of mammography, MRI, and ovarian ultrasound examinations undergone during the previous three years. The same questions were also asked about mammography and ovarian ultrasound at the one and two year follow-ups but the question about MRI was asked only at the five-year follow-up, since the medical guidelines only recommended this type of investigation for the first time in 2004 [START_REF] Eisinger | Recommendations for medical management of hereditary breast and ovarian cancer: the French National Ad Hoc Committee[END_REF][START_REF] Eisinger | Identification and management of hereditary predisposition to cancer of the breast and the ovary (update 2004)[END_REF] . The occurrence of prophylactic surgery was cross-checked with the medical records. Details about prophylactic surgery (RRM, RRSO) were collected prior to disclosure of results and then again at the one-, two-and five-year follows. Baseline preventive behaviours had been asked in the pre-disclosure questionnaire.

Perception of personal risk of cancer.

Perception of the personal risk of developing breast cancer was measured on a six-point Likert scale before disclosure and at follow-up by asking the same question: "Do you think your risk of developing breast cancer is: "very high" "high" "moderate" "low" "very low" "don't know". The same questions were asked about respondents' perception of their risk of developing ovarian cancer and about their perception of the risk of developing cancer of other kinds.

Socio-demographic, psychological and medical characteristics

Socio-demographic variables such as age, number of children, level of education, occupation, prior preventive behaviour and psychological variables were collected at baseline for all the women included and updated during follow-up.

The medical characteristics included the number of first and second degree relatives affected with breast/ovarian cancer at inclusion, and the deleterious BRCA1/2 mutation involved.

Statistical Analysis

Chi-square tests, t-tests, and ANOVA were used to make univariate comparisons on categorical and continuous variables. McNemar's chi-square tests were used to compare respondents' cancer risk perceptions at different times. The level of statistical significance (α error) was taken to be p≤0.05.

Data analysis was conducted in 3 stages.

-To check how representative the final sample was after 5 years, the respondents' and non-respondents' baseline sociodemographic, psychosocial and medical characteristics were compared.

-The preventive strategies used by carriers/non carriers were recorded at the 5-year follow-up. They are presented in a decision tree and the two groups were then compared after stratifying the results by age-group. The sequences of preventive options chosen by BRCA1/2 carriers were analysed at the 5-year follow-up.

-High/very high breast, ovarian, and other cancer risk perceptions documented at various times were studied, depending on the preventive strategies chosen (or not) by carriers/non carriers. High and very high risk perception levels were grouped together and denoted "high" perceptions.

The Statistical Package for Social Science (SPSS/PC Released 17.0) was used to perform all the statistical analyses.

RESULTS

Respondents vs non respondents

Among the 345 women who completed the first questionnaire before test result disclosure and who should have reached the five years follow-up endpoint, 14 were excluded because they had developed breast cancer (n=12) or ovarian cancer (n=2) by the 5 th year of followup; all 14 were BRCA1/2 mutation carriers. Among the unaffected women at five years (N=331), 246 (74%) completed all five questionnaires and were included in this analysis.

Respondents and non-respondents did not differ significantly in their baseline sociodemographic, psychological or medical characteristics. However, respondents had reported doing Breast Self Examination significantly more frequently than non-respondents (p≤0.05) in the baseline questionnaire.

Sample Characteristics

Among these 246 women, 41% (N=101) were carriers of a familial BRCA1/2 mutation, whereas 59% (N=145) were non-carriers. The mean age of the BRCA1/2 carriers at the time of disclosure was 37.2 years (SD=10.2), which differed significantly from that of the non-carriers (41.7 ± 11.8) (±SD) (p=0.002). The level of education, marital status and number of children did not differ significantly between BRCA1/2 mutation carriers and noncarriers. Medical characteristics such as the number of first degree relatives affected were also similar between carriers and non-carriers. Respondents' characteristics are given in Table 1. The 246 respondents belonged to 166 different families: 68.1% (n=113) of these families were represented in the sample by a single woman, whereas the remaining, 31.9% (n=53) were represented by several members, numbering 2.5 on average (median 2; range 2 to 6). Among the 18 centres who reported carriers, 3 had reported 56.5% of the cases analysed (n=57) and 15 had reported the remaining 44.5% (n=44). The average number of carriers by centre was 5.6 (range 1-31).

Preventive strategies in mutation carriers/non-carriers after disclosure of the BRCA1/2 test results

Carriers (n=101): a decision tree was drawn up describing the seven strategies documented at the five-year follow-up, including prophylactic surgery and breast imaging (Figure 1). Since Transvaginal Ultrasound is not thought to be an effective [START_REF] Evans | Screening for familial ovarian cancer: poor survival of BRCA1/2 related cancers[END_REF] All those who eventually opted for RRM by the 5th year of follow-up (Strategies S1 and S2

in figure 1; n=7) had high breast cancer risk perceptions before disclosure; these high perceptions decreased with time after disclosure and after undergoing RRM (Figure 3).

High breast cancer risk perceptions increased steadily with time among all the other carriers, regardless of whether or not they opted for Risk Reducing Salpingo-Oophorectomy (Strategies S3 and S4 in figure 1; n=38) or not (Strategies S5-S7, figure 1; n=56).

High ovarian cancer risk perceptions with time showed the same pattern as with breast cancer. Among carriers who did not opt for RRM, those who opted for RRSO (Strategies S3-S4 in Figure 1) had higher ovarian cancer risk perceptions before disclosure than those who did not (Figure 4; p=0.014). Between 6 and 60 months after disclosure of the results, these high perceptions decreased significantly after RRSO (McNemar; p=0.002), whereas a significant increase (McNemar; p=0.049) occurred among those who did not opt for surgery.

Discussion

This is the first time the preventive strategies used by a French national cohort of unaffected female BRCA1/2 carriers and non carriers have been documented for five years after routine genetic test result disclosure. The preventive strategies reported by respondents five years after disclosure show that in France, regular MRI surveillance (along with other breast imaging methods) is the most frequently preferred strategy among unaffected female BRCA1/2 carriers under 40 years of age, whereas RRSO tends to be combined with regular MRI breast imaging among older women (Figures 2a,2b). When the decision to undergo RRM has been made, RRSO is generally undergone as well (Figure 1).

Trans-vaginal ultrasound investigations were undergone regularly by carriers without RRSO, and non-carriers appeared to favour opting for screening despite their low cancer risk (table 2). In France this screening is possible when prescribed by a medical practitioner. High breast/ovarian cancer risk perceptions were significantly affected in both the short and long term among carriers and non-carriers alike. In the long run, high breast cancer risk perceptions increased steadily with time among those without RRM (and decreased among the small sample with RRM), and a similar pattern was observed in the case of ovarian cancer risk perception and RRSO. RRSO had no effect on breast cancer risk perceptions.

It was reported some time ago that there is little enthusiasm in France for RRM, either among health professionals or women attending cancer genetic clinics [START_REF] Julian-Reynier | Physicians' attitudes towards mammography and prophylactic surgery for hereditary breast/ovarian cancer risk and subsequently published guidelines[END_REF][START_REF] Bouchard | Women's attitudes toward preventive strategies for hereditary breast or ovarian carcinoma differ from one country to another: differences among English, French, and Canadian women[END_REF] . The present results confirm the reluctance previously observed about these issues, although the rate of uptake of RRSO in France is comparable to that of other countries in the relevant agegroups. Health professionals in France seem to expect MRI to be a highly effective means of detecting early forms of breast cancer, resulting in a good prognosis, whereas those in other countries prefer the highly effective surgical strategies available. It would be interesting to know the survival rates associated with each combination of preventive strategies, which would help to give women all the information required for their decisionmaking to be autonomous and well-informed.

It is worth discussing the increase observed here in the high breast/ovarian cancer risk perceptions of those who did not undergo risk reducing surgery, as well as the fact that RRSO did not decrease high breast cancer risk perceptions although this intervention is known to significantly decrease the risk of breast cancer [START_REF] Rebbeck | Meta-analysis of risk reduction estimates associated with risk-reducing salpingo-oophorectomy in BRCA1 or BRCA2 mutation carriers[END_REF] . Finch et al. [START_REF] Finch | Breast and ovarian cancer risk perception after prophylactic salpingo-oophorectomy due to an inherited mutation in the BRCA1 or BRCA2 gene[END_REF] retrospectively assessed the impact of genetic counseling and RRSO on breast and ovarian cancer risk knowledge and concluded that most women accurately perceived their breast cancer risks.

Our results mean either that the information was not delivered by health care professionals or that RRSO had no impact on a priori lay beliefs about breast cancer risks. Cancer risk perception is a complex and subjective issue, since risk perception seems to have nothing to do with "risk knowledge". Risk perception is known to determine behavior, whereas people's "risk knowledge" is not predictive of their health-related behavior [START_REF] Julian-Reynier | Risk communication strategies: state of the art and effectiveness in the context of cancer genetic services[END_REF] . Risk perception may decrease when the main target organ of cancer has been removed surgically, whereas the situation is not the same when another organ is taken out, and the preventive action obtained through the suppression of hormonal effect. Previous studies have shown that women's psychological distress decreases after undergoing prophylactic mastectomy [START_REF] Metcalfe | Psychosocial functioning in women who have undergone bilateral prophylactic mastectomy[END_REF] , and that their fear of developing cancer also decreases, [START_REF] Van Oostrom | Long-term psychological impact of carrying a BRCA1/2 mutation and prophylactic surgery: a 5-year follow-up study[END_REF] which is similar to what occurs with high risk perception or affective risk perception [START_REF] Van Dooren | Psychological distress in women at increased risk for breast cancer: the role of risk perception[END_REF] . However, other authors have reported that the levels of distress are still high after these interventions, especially RRSO [START_REF] Bresser | The course of distress in women at increased risk of breast and ovarian cancer due to an (identified) genetic susceptibility who opt for prophylactic mastectomy and/or salpingo-oophorectomy[END_REF] , possibly because these women's perception of the remaining risk of breast cancer has not changed significantly.

Our study had several limitations. First, respondents always differ from non-respondents, even if they have the same baseline characteristics. Only baseline Breast Self Examination practices differed here between respondents and non-respondents; therefore we must remember that our results may not be completely representative of the whole cohort or of the whole French population of BRCA1/2 carriers, although we benefited from the advantages of multicentre, nation-wide recruitment. Secondly, the same bias occurred as in previous studies, since following the same group of people with questionnaires may affect their spontaneous decisions during follow-up and hence the methods of surveillance and prevention they choose. We also have to take the time-factor into account, since preventive recommendations evolve with time and while the people in this study were being tested, new preventive recommendations were issued in 2004, especially as regards prophylactic surgery, which was more strongly recommended [START_REF] Eisinger | Identification and management of hereditary predisposition to cancer of the breast and the ovary (update 2004)[END_REF] than previously [START_REF] Eisinger | Recommendations for medical management of hereditary breast and ovarian cancer: the French National Ad Hoc Committee[END_REF] .

In conclusion, women's cancer risk perceptions five years after disclosure of their BRCA1/2 status depend on decisions about preventive interventions more than simply on the outcomes of mutation tests . Women need to know more these days about the probability of survival associated with all the strategies available, and in particular whether opting for RRSO and breast MRI surveillance is likely to be as effective as undergoing both RRM and RRSO in terms of survival.

words

Acknowledgements:

All the members of the Genepso cohort investigators including, Claude Adenis, Yves-Jean Roxane Fabre and Anne-Deborah Bouhnik are acknowledged for their statistical expertise.

Financial support:

Grant from the "Institut National du Cancer" (National Cancer Institute) R08097AA/RPT08011AAA INCA

Figure 1 :Figure 3 :Figure 4 :

 134 Figure 1: Preventive management strategies observed in BRCA1/2 mutation carriers (N=101) five years after test result disclosure

Figure 1 :Figure 2a :

 12a Figure 1: Preventive management strategies observed in BRCA1/2 mutation carriers (N=101) five years after test result disclosure

1

  from RRM **: apart from RRSO Mean number of investigations undergone during the last 3 years (means and standard deviations)

High perceptions of breast/ovarian cancer risks versus preventive strategies and mutation carrier/non carrier status.

  means of improving ovarian cancer prognosis and is not officially recommended in France[START_REF] Eisinger | Recommendations for medical management of hereditary breast and ovarian cancer: the French National Ad Hoc Committee[END_REF][START_REF] Eisinger | Identification and management of hereditary predisposition to cancer of the breast and the ovary (update 2004)[END_REF] , it was not included in these decision trees but noted separately. Four strategies accounted for 88% of the preventive options chosen by the carriers at the end of the five year follow-up: MRI was undergone by 63% (in 59/94 cases without RRM), and no correlations were observed with age in this respect. A high rate of ovarian ultrasound screening was still observed during the last 3 years among those who underwent no RRSO. The results obtained on each age-group are presented in Table2.

	Non-carriers (n=145): No RRM was performed on non-carriers after disclosure of the
	results, but 3 Salpingo-Oophorectomies were undergone by non-carriers for other medical
	reasons (fibroma or cystic ovaries) (Table 2). 53% of the women aged between 30 and 39
	years (20% of the <30 age-group) reported at the final follow-up that they had continued to
	undergo annual or two-yearly mammography during the previous 3 years; only 7 (4.8%)
	had undergone MRI; 43.2% stated that they had undergone an ovarian ultrasound
	examination during the last 3 years.
	Disclosure of the BRCA1/2 status significantly
	affected the carriers and non carriers' high perceptions of breast/ovarian cancer risks.
	Among the carriers, it significantly increased the proportion of carriers with high/very high
	breast cancer risk perceptions (from 63% before disclosure to 81% 6 months after
	disclosure, McNemar's chi-square, p=0.016) and some carriers' high ovarian cancer risk
	perceptions (from 42% before disclosure to 62% 6 months after disclosure, McNemar's chi-
	square p=0.007); whereas it decreased high breast cancer risk perceptions (N=145) (figure
	3; p<0.001) and high ovarian cancer risk perceptions (figure 4; p<0.001) in non-carriers.
	High perception of other cancer risks was relatively infrequent and not affected by
	Among the carriers, RRM was undergone by 7%, and this option was not correlated with disclosure (12.1% and 9.5% before disclosure and 6 months after disclosure, respectively,
	the women's age. RRSO was undergone by 43% of the carriers, and this option was McNemar's chi-square p=0.248 and 11.2%, 7.9%, and 15.5% after 1, 2 and 5 years,
	chosen significantly more frequently with age (p<0.001): the rate of uptake increased from respectively).

50% had opted for breast surveillance alone, based on either Magnetic Resonance Imaging (MRI) and other imaging methods in 31% of cases (n=31; strategy S5), or based on mammography alone in 19% (n=19; S6); 38% had opted for Risk Reducing Salpingo-Oophorectomy and breast surveillance based on MRI and/or other imaging methods in 28% of cases (n=28; S3) or mammography alone in 10% (n=10; S4). The other three strategies documented were Risk Reducing Mastectomy (RRM) and RRSO (5%) (n=5; S1), RRM alone (n=2; 2%; S2), and neither RRM/RRSO nor surveillance (n=6; 6%; S7). One 45-year old woman who had opted for S2 planned to undergo RRSO 5 weeks after completing the final questionnaire; the other one was 48 years old and had no family history of ovarian cancer. In the S7 group, 2 women were under 30 years of age; the others were aged between 31 and 39. These strategies were stratified by age, above and below 40 years of age, as shown in figures 2a and 2b.

18% in the 30-to 39 year -olds to 65% in those ≥ 40 years of age. During the last 3 years, the women underwent 2.8 mammograms on average and 1.7 MRI on average (table 2).

Table 1 :

 1 Baseline medical and socio-demographic characteristics of study population(N= 246)

	Carriers	Non carriers
	N=101		N=145
	n	%	n	%	p*

Table 2 :

 2 Preventive strategies adopted by BRCA1/2 carriers and non carriers (all unaffected) five years after test result disclosure (N=246)

	Carriers (N=101)	Non-Carriers (N=145)


Rabah Ben Yaou

Claire L Navarro

Susana Quijano-Roy

Anne T Bertrand

Catherine Massart

Annachiara De Sandre-Giovannoli

Juan Cadiñanos

Kamel Mamchaoui

Gillian S Butler-Browne

Brigitte Estournet

Pascale Richard

Annie Barois

Nicolas Levy

Gisèle Bonne
email: g.bonne@institut-myologie.org

Type B mandibuloacral dysplasia with congenital myopathy due to homozygous ZMPSTE24 missense mutation

Keywords: ZMPSTE24, Mandibuloacral dysplasia, Congenital myopathy, prelamin A, Secondary laminopathies

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

ZMPSTE24 (also known as FACE-1) encodes a ubiquitously expressed zinc metalloprotease. Prelamin A, the lamin A precursor, is the only known substrate of ZMPSTE24 in mammals. [START_REF] Kilic | Subcellular localization and partial purification of prelamin A endoprotease: an enzyme which catalyzes the conversion of farnesylated prelamin A to mature lamin A[END_REF] ZMPSTE24 is involved in post-translational proteolytic cleavage of carboxy terminal residues of farnesylated prelamin A to form mature lamin A. [START_REF] Leung | Biochemical studies of Zmpste24-deficient mice[END_REF] Prelamin A is initially farnesylated on the last cystein within the prenylation "CaaX" motif at the Cterminus end of the protein, by a farnesyl-transferase. [START_REF] Lutz | Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina[END_REF] Then, ZMPSTE24 or RCE1 [START_REF] Varela | Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging[END_REF] removes the last three residues "aaX", before a carboxy-methyl group is added by the methyl transferase ICMT on the last remaining cystein. [START_REF] Sinensky | The processing pathway of prelamin A[END_REF] Finally, a second cleavage is specifically performed by ZMPSTE24, removing the last 15 amino acids. [START_REF] Corrigan | Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24[END_REF] The product of this posttranslational maturation pathway, mature lamin A, is located both at the nuclear lamina and the nucleoplasm. A-type lamins A and C, are expressed in all vertebrate differentiated cells, and are translated from alternatively spliced transcripts of the LMNA gene. After posttranslational modifications A-type lamins assemble with B-type lamins to form the nuclear lamina, a filamentous meshwork underlying the inner nuclear membrane. [START_REF] Broers | The nuclear lamins: laminopathies and their role in premature ageing[END_REF] While LMNA mutations are known to cause several phenotypes named "laminopathies" (OMIM *150330) involving striated muscles, adipose, nervous, cutaneous tissues and bone in isolated or combined fashions, [START_REF] Broers | The nuclear lamins: laminopathies and their role in premature ageing[END_REF][START_REF] Worman | Laminopathies": a wide spectrum of human diseases[END_REF] the responsibility of ZMPSTE24 gene mutations (OMIM *606480) in human disease has been also recognized. By a candidate gene strategy resulting from phenotype similarities, various homozygous or compound heterozygous ZMPSTE24 mutations were identified in two different and extremely rare premature ageing disorders: restrictive dermopathy (RD, OMIM #275210) and B type mandibuloacral dysplasia (MADB, OMIM #608612). [START_REF] Agarwal | Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia[END_REF][START_REF] Navarro | Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganisation and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors[END_REF] RD, the most severe disorder, is a lethal neonatal genodermatosis [START_REF] Witt | Restrictive dermopathy: a newly recognized autosomal recessive skin dysplasia[END_REF] characterized by intrauterine growth retardation, tight, rigid and easily eroded skin with prominent superficial vessels, bone mineralization defects, dysplastic clavicles, arthrogryposis and early neonatal death. MADB, an autosomal recessive disorder, characterized by skeletal abnormalities including mandible and clavicle hypoplasia, acro-osteolysis of the terminal phalanges, mottled pigmentation, cutaneous atrophy and generalized lipodystrophy with metabolic syndrome such as insulin resistance, glucose intolerance, diabetes mellitus and hypertriglyceridemia. [START_REF] Novelli | Mandibuloacral dysplasia is caused by a mutation in LMNA encoding lamins A/C[END_REF] In both disorders, no skeletal muscle involvement was reported so far.

The lack of ZMPSTE24 protein or its activity in cell lines from ZMPSTE24 mutated patients resulted in misshapen nuclei containing abnormally aggregated lamin A/C associated with accumulation of unprocessed prelamin A. [START_REF] Navarro | Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganisation and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors[END_REF][START_REF] Shackleton | Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype[END_REF][START_REF] Denecke | A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS[END_REF][START_REF] Sander | A newly identified splice site mutation in ZMPSTE24 causes restrictive dermopathy in the Middle East[END_REF] Whereas the ZMPSTE24 mutations found in MADB encoded a protein with considerable reduced residual activity, the mutations found in RD led to null ZMPSTE24 activity strongly suggesting a positive correlation between residual enzyme activity, accumulated prelamin A levels and the severity of the phenotype. [START_REF] Smigiel | Novel frameshifting mutations of the ZMPSTE24 gene in two siblings affected with restrictive dermopathy and review of the mutations described in the literature[END_REF] In Zmpste24 knockout mice, severe growth retardation, skeletal abnormalities including small mandible, lipodystrophy, cardiomyopathy, muscular dystrophy were reported as well as premature death, these features being associated to a complete absence of mature lamin A and an exclusive production of prelamin A. [START_REF] Pendas | Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice[END_REF][START_REF] Bergo | Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect[END_REF] More importantly, several studies have demonstrated that blocking prelamin A farnesylation using either farnesyl transferase inhibitors (FTI) or a combination of statins and aminobisphosphonates could prevent prelamin A accumulation, extend longevity and ameliorate disease phenotype in Zmpste24 -/-mice [START_REF] Varela | Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging[END_REF][START_REF] Fong | A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria[END_REF] leading to therapeutic trials in diseases associated to defective prelamin A processing.

Here, we report a 30 year longitudinal follow up of a patient carrying a new homozygous missense ZMPSTE24 mutation leading to partial loss of enzymatic activity. The resulting clinical phenotype goes beyond the expected progeroid features and includes skeletal muscle involvement, suggesting that defective prelamin A processing may also induce muscle disease.

PATIENTS AND METHODS

Patient clinical history was assembled from the detailed medical reports of the neuropediatric department, Raymond Poincarré hospital, Assistance Publique Hôpitaux de Paris (AP-HP), Garches, France, where the patient was followed along her whole life. Our study complies with the ethical guidelines of all the institutions involved. Genetic analysis were performed on DNA samples from peripheral blood lymphocytes of the patient, her non affected parents, two sisters and one brother after informed consent.

LMNA and ZMPSTE24 gene screening was performed as described previously. [START_REF] Navarro | Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganisation and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Yaou | Multitissular involvement in a family with LMNA and EMD mutations: Role of digenic mechanism?[END_REF] Confirmation of ZMPSTE24 mutation (c.281T>C) was performed by enzymatic digestion of the PCR product using BslI (Ozyme, Saint-Quentin-en-Yvelines, France). ZMPSTE24 conformations were predicted in silico using the two algorithms TMPred (http://www.ch.embnet.org/software/TMPREDform.hml) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Primary skin fibroblasts were obtained from cultured skin biopsies of the patient and a normal control subject according to Muchir et al. [START_REF] Muchir | Nuclear envelope alterations in fibroblasts from patients with muscular dystrophy, cardiomyopathy, and partial lipodystrophy carrying lamin A/C gene mutations[END_REF] Western blot analysis was performed from 40μg of protein homogenates on 10% SDS-PAGE for both control and patient primary skin fibroblasts. Detection of ZMPSTE24 and lamins A and C was performed using monoclonal anti-ZMPSTE24 antibody (205-8C10, Daiichi Chemical) [START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors[END_REF] and polyclonal anti-lamin A/C antibody (H-110, Santa Cruz Biotechnologies, CA, USA). [START_REF] Yaou | Multitissular involvement in a family with LMNA and EMD mutations: Role of digenic mechanism?[END_REF] Immuno-fluorescence analysis of lamins A/C or prelamin A were then performed using polyclonal anti-lamin A/C (both at 1/100, Santa Cruz Biotechnologies) and FITC-conjugated anti-rabbit (1/500, Santa Cruz Biotechnologies) or Alexa-568 anti-goat (1/500, Invitrogen, USA) while emerin was detected by monoclonal MANEM5 anti-emerin (1/50, from G. Morris, NEWI, UK) and Alexa-568 anti-mouse (1/500, Invitrogen, USA).

Life span analysis of fibroblasts from the patient and an age-matched healthy control was performed as previously described. [START_REF] Decary | Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy[END_REF] Cell populations were serially passaged until they stopped dividing. At cell isolation, all cell populations were considered to be at 1 mean population doubling (MPD). The number of population doublings at every passage was calculated as log (N/n)/log 2 where N is the number of cells at the time of passage divided by the number of cells initially seeded (n).

RESULTS

Clinical phenotype

The patient is the 4 th female born to second degree related Portuguese parents. There were no other antecedents in the family. Hydramnios was detected intra-utero and delivery was premature at 35 weeks of gestational age. Birth weight was 1.715 kg (-3 SD), length 45 cm (-4 SD) and head circumference of 29.5 cm (-4SD) indicating intrauterine growth retardation.

Apgar test was normal but two episodes of cyanosis were noted during the 1 st day of life. Joint contractures were observed, including ankles, knees (popliteal angle=90°), hips and elbows.

Skin was dysmature, with atrophic, dried, crumpled, and desquamated hyperkeratotic aspect.

Dysmorphic features with retrognathia were also associated. Karyotype was normal.

Polycythemia (hematocrit=63.1%) and thrombocytosis (thrombocyte count=514000/mm3) were also present at birth. Although mild axial and limb hypotonia was noted by the age of 3 months (mo), motor milestones were within normal ranges. She walked independently at 17mo. Difficulties to walk, climb stairs or rise from the floor appeared by the age of 2 years (y). Electromyography revealed myopathic patterns on deltoid, flexor carpi radialis and tibialis anterior muscles without spontaneous activities. Nerve conduction velocities were normal. Muscle weakness predominant in peri-scapular regions and proximal lower limbs progressed particularly from the age of 4y. There was scapular winging (figure 1, a 3-5 ). A deltoid muscle biopsy performed at 9y revealed myopathic changes suggestive of fibre type disproportion. There was fibre size variation with a large number of small fibres and type I fibre predominance (figure 1,d 1 ). All features were suggestive of a congenital myopathy with fibre type disproportion. In the second decade, muscle deficit spread to the 4 limbs. At 26y muscle weakness extended rapidly to distal lower limbs and electromyography examination revealed peroneal nerve involvement in addition to a myopathic pattern at needle electromyography.

The patient demonstrated a severe failure to thrive (figure 1a), that led to a 'harmonic' dwarfism (figure 1, a 1-5 ). Varied and severe complications were observed during the course of the disease involving bones, skin, joints, skeletal, respiratory and cardiac muscles, and vascular, renal and hormonal systems. However, cognition was normal, she attended school, drove an adapted car and worked as an accountant until 25y.

Bone involvement was an early complication. Hypoplasia of the mandibula (figure 1,c Serum calcium, phosphate and alkaline phosphatase levels were normal since birth.

Cutaneous abnormalities typical of progeria such as reduced subcutaneous fat were noted since birth. Generalized sclerodermic skin and abdominal papules were observed during the first year (figure 1, a 2 ). By 14mo, frontal and abdominal veins were dilated (figure 1,a 3 ) and the abdominal papules became beige and translucent. Later, blue spots appeared on the face and legs and a skin biopsy performed at 9y revealed sclerodermic features in the absence of deposits or inflammatory infiltrates. There was extreme dermal fibrosis, increased and often horizontally oriented elastic fibres, basal lamina horizontalisation and rare dermal papules (data not shown). Subcutaneous calcifications appeared in different sites (inter-phalangian joints, cervical, dorsal and thoracic, figure 1, c 8-9 ). From 13.5y there was a rapid loss of hair evolving to alopecia in the year after (figure 1, a 6 ).

The cervical paravertebral calcifications caused spine and chest rigidity and other cervical subcutaneous nodules probably were responsible for swallowing difficulties observed at 15y. By 26y loss of muscle strength was predominant in lower limbs and a vertebral CT scan identified multiple subcutaneous and paravertebral calcifications at the cervicothoracic levels, infiltrating the left C7-T1 intervertebral foramen, the ribs and pushing back the scapula. Later, vertebral MRI and CT-scan revealed 2 intracanalar calcifications, one reducing medullar cavity diameter and another causing medullar compression and infiltrated the intervertebral foramen. Surgical ablation of these calcifications led to good functional recovery. Joint contractures associated with the short clavicles resulted in chest flatness with protuberant right chondro-costal joints. Joint mobility became very limited during the course of the disease at the ankles, knees and hips and a marked limitation of the mobility of shoulders and contractures of pectoral muscles was observed. With respect to the spine, by 2y a mild dorsal kypho-scoliosis was noted and associated cervical and lumbar lordosis due to axial weakness.

A Milwaukee brace was indicated at 6y. By 15.5y, a dorsal gibbus was reported.

Respiratory complications were a late event but the patient died as a consequence of them. At 8y, diurnal hypersomnia, dyspnea, nocturnal snoring and an impressive laryngeal stridor prompted the first respiratory evaluation. Forced vital capacity (FVC) was reduced to 40% of theoretical value, with a significant fall in supine FVC with respect to sitting position, indicating a selective diaphragmatic failure and a major respiratory difficulty when lying.

Polysomnographic evaluation revealed nocturnal apneas and marked desaturation. The patient was treated by non invasive noctural ventilation. Theophylline was also started. Restrictive respiratory insufficiency was observed (FVC of 37% at 10y and 28.2% at 25y).

ECG and echocardiography were normal until 24y when sinusal tachycardia, sharpened T waves, right bundle branch block, ventricular extrasystols and concentric left ventricular hypertrophy as well as high blood pressure were observed. At 27y, blood renin (34.9 pg/ml, N<19) and aldosterone (207 pg/ml, N<125) were elevated concomitantly with hypokalaemia (3.3 mmol/l, N>3.5). This prompted a CT-scan of the renal arteries as well as MRI coupled with angiography and revealed bilateral calcifications of the renal arteries (figure 1,d

2 )
resulting in a 50% reduction in diameter without renal parenchyma vascularisation abnormalities. Atheromatous aortic infiltration was also observed (figure 1,d 3 ). High blood pressure was treated by acebutolol and enalapril with no significant improvement. At 29y, chronic kidney disease with severe anaemia appeared and worsened progressively. Non-insulin dependent diabetes mellitus was diagnosed but no treatment was administered. The last evaluation at 30y showed elevated serum urea (23 mmol/l, N<7.5), creatinine (455 µmol/l, N<120) and phosphate (2.73 mmol/l, N<1.5) levels, the glomerular filtration rate was 9 ml/mn/1.73m 2 (N>80), proteinuria (6.9 g/l), glycosuria (2.5 g/l) and beta2 microglobinuria (25000 mg/24h). MAG3 scan revealed a global reduction in tracer uptake and excretion; all suggestive of multifactor chronic kidney disease with glomerular, vascular and tubular abnormalities. Due to bilateral renal artery calcifications, a revascularization procedure was suggested. Meanwhile, haemodialysis as well as chronic peritoneal dialysis were also considered. Subsequently, at 30 years of age, in this context of severe renal insufficiency and high blood pressure, the patient developed a lung infection. During this episode; she felt unwell, went to bed and was found dead in her bed at home. No autopsy was performed.

Further investigations have been performed. Blood testing revealed additional abnormalities during the disease. At 17mo, pre-betalipoproteins were slightly elevated (6.2 g/L, N<6). At 12y slight hearing loss was suspected at school. However, audiogram and auditory evoked potentials remained normal. Puberty was acquired at 12.5 y without menarche or menstrual period anomalies. The patient had poor breast development. Hormonal evaluation at 13y, revealed reduced dehydroepiandrosterone (1.6 ng/ml, N>2) and estradiol (27pg/ml, N>60), elevated somatostatin (28 pg/ml, N<10), with the remaining hormonal parameters remaining normal (cortisol, deoxycortisols, progesterone, androstenedione, testosterone, corticotropin and prolactin). At 26.5y, hypertriglyceridemia (3.65 mmol/l, N<1.69) hypercholesterolemia (7.54 mmol/l, N<5.2) hypertransaminemia (ALAT: 201 IU/L, N<35; AST: 93 IU/L, N<34) and increased gamma-GT (244 IU/L, N<28) was observed.

Abdominal ultrasonography revealed global hepatomegaly with steatosis with normal and symmetric ovaries. Medical treatment with simvastatin was started. CK were always within the normal range.

Genetic analysis

Because the main clinical features suggested a progeroid syndrome, with clinical features similar to MAD or HGPS, LMNA was first sequenced at the genomic level but no mutation was found in the exons or intron/exon boundaries. The ZMPSTE24 gene was subsequently sequenced and a homozygous variation (c.281T>C) in exon 3, predicted to result in p.Leu94Pro was identified (figure 2a). This variation creates a new BslI restriction site that was further used to confirm the mutation in the patient and analyse its segregation within the family. A heterozygous c.281T>C variation was identified in the non affected parents, one sister and the brother, while it was absent in the other sister (figure 2b). This variation was not detected in more than 200 control chromosomes from unrelated subjects. Partial alignment of the ZMPSTE24 protein sequences from various species showed that the mutated Leucine 94 is highly conserved, suggesting an important role of this residue in the enzymatic activity (figure 2c). In silico predictions using TMHMM and TMpred programs, with either normal or mutated ZMPSTE24 protein sequences as matrix, indicated two possible conformational models: one corresponded to the normally folded protein including its seven consensus transmembrane (TM) domains, whereas the other lacked the second TM (figure 2d).

Consequently this misfolded ZMPSTE24 predicted both its catalytic and endoplasmic reticulum (ER) signal mislocated in the ER lumen instead of cytoplasm, suggesting a loss of function or a mislocalisation. These predictions could not be considered as being mutually exclusive.

Western blot analysis

To determine whether or not these ZMPSTE24 variations modify the expression of ZMPSTE24 protein, and consequently to prelamin A processing defects, western blot analyses were carried out on proteins extracted from the patient's cultured skin fibroblasts at passage P2. Immunoblots on the patient's fibroblast revealed an important decrease in the level of ZMPSTE24 compared to age matched control fibroblasts. As expected, this reduction was associated with the accumulation of prelamin A: in addition to the wild-type lamins A and C, an extra-band with higher molecular weight at the expected prelamin A size was observed (figure 2e). This supplementary band was absent from control fibroblasts also analysed at passage P2.

Life span and expression of prelamin A, lamin A/C and emerin of fibroblasts

The patient's fibroblasts were highly variable in size with a majority of the cells being larger than control cells. They tended to grow as clusters (figure 3) and exhibited a reduced life span (figure 4). Primary patient fibroblasts arrest their growth after only 56 days in culture (7 divisions). This is in contrast to primary control fibroblasts which made 38 divisions after 130 days in culture (figure 4). Patient fibroblasts exhibited major nuclear shape defects.

Although lamins A/C and emerin were still localised at the nuclear rim together with prelamin A in all patient's fibroblasts, severe nuclear shape defects (lobulations, herniations) were observed, as compared with the uniform oval or round nuclei in the wild-type cells (figure 3).

DISCUSSION

This is the first report describing a MADB phenotype associated to myopathic features caused by a homozygous missense ZMPSTE24 mutation. The pathogenicity of this unreported mutation was demonstrated by the presence of both prelamin A in protein extracts from patient fibroblasts, the abnormal shape of the fibroblast nuclei and the reduced lifespan of these fibroblasts.

In the literature, 30 patients carrying ZMPSTE24 mutations have already been reported and have a large clinical spectrum going from typical RD to MADB. Apart from one, [START_REF] Thill | Restrictive dermopathy: a rare laminopathy[END_REF] the 23 patients with typical RD features had either homozygous or compound heterozygous null mutations (nonsense, insertions or deletions) [START_REF] Navarro | Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganisation and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors[END_REF][START_REF] Smigiel | Novel frameshifting mutations of the ZMPSTE24 gene in two siblings affected with restrictive dermopathy and review of the mutations described in the literature[END_REF][START_REF] Moulson | Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy[END_REF][START_REF] Morais | Restrictive dermopathy--a lethal congenital laminopathy. Case report and review of the literature[END_REF][START_REF] Kariminejad | Restrictive dermopathy. Molecular diagnosis of restrictive dermopathy in a stillborn fetus from a consanguineous Iranian family[END_REF][START_REF] Chen | A case of restrictive dermopathy with complete chorioamniotic membrane separation caused by a novel homozygous nonsense mutation in the ZMPSTE24 gene[END_REF][START_REF] Jagadeesh | Prenatal diagnosis of restrictive dermopathy[END_REF][START_REF] Li | Homozygosity for the common mutation c.1085dupT in the ZMPSTE24 gene in a Mennonite baby with restrictive dermopathy and placenta abruption[END_REF] or very large in-frame deletions sometimes resulting from splice site mutations [START_REF] Navarro | Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganisation and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors[END_REF][START_REF] Sander | A newly identified splice site mutation in ZMPSTE24 causes restrictive dermopathy in the Middle East[END_REF] in the ZMPSTE24 gene. Moreover, all RD related mutations lead to a complete absence of the ZMPSTE24 protein or to null activity, consequently causing the accumulation of prelamin A and an absence of mature lamin A. [START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors[END_REF][START_REF] Sander | A newly identified splice site mutation in ZMPSTE24 causes restrictive dermopathy in the Middle East[END_REF][START_REF] Moulson | Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy[END_REF] Two patients carrying ZMPSTE24 mutations had progeroid features of variable severity. The first one, carrying compound nonsense and missense mutations, had been diagnosed as a severe progeroid phenotype and died at 2y. [START_REF] Shackleton | Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype[END_REF] The second one, carried a homozygous ZMPSTE24 frameshift deletion associated to a LMNA heterozygous nonsense mutation which "rescued" the patient's phenotype, most probably by reducing to half the amounts of accumulated prelamin A, as in the double knock out Zmpste24 -/-Lmna +/-mice. [START_REF] Denecke | A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS[END_REF][START_REF] Fong | Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice[END_REF] This patient showed features compatible with a severe form of MADB, but was described as being affected with HGPS. [START_REF] Denecke | A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS[END_REF] In this last report, the authors concluded that the LMNA truncation is a salvage alteration modifying the clinical picture from RD to HGPS by preventing farnesylation of the accumulated prelamin A or by reducing the amounts of the accumulated prelamin A. This second hypothesis is more probable from our point of view, since no truncated lamin A isoform was visible on the western blot. In all cases, farnesylated prelamin A accumulated while mature lamin A was either absent [START_REF] Denecke | A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS[END_REF] or reduced. [START_REF] Shackleton | Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype[END_REF] Finally, 5 patients carrying compound heterozygous nonsense and missense ZMPSTE24 mutations, have been described as being affected with MADB. [START_REF] Agarwal | Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia[END_REF][START_REF] Agarwal | Focal segmental glomerulosclerosis in patients with mandibuloacral dysplasia owing to ZMPSTE24 deficiency[END_REF][START_REF] Miyoshi | Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings[END_REF][START_REF] Cunningham | Skeletal phenotype of mandibuloacral dysplasia associated with mutations in ZMPSTE24[END_REF] These ZMPSTE24 mutations result in the presence of traces of prelamin A but also to mature lamin A [START_REF] Miyoshi | Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings[END_REF] due to partial residual ZMPSTE24 activity. [START_REF] Agarwal | Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia[END_REF][START_REF] Agarwal | Focal segmental glomerulosclerosis in patients with mandibuloacral dysplasia owing to ZMPSTE24 deficiency[END_REF][START_REF] Miyoshi | Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings[END_REF] All these reports strongly suggest a link between the phenotype severity, prelamin A accumulation levels and ZMPSTE24 residual activity. Complete prelamin A accumulation with null ZMPSTE24 enzyme activity can be correlated to the most severe progeroid syndrome, RD; null ZMPSTE24 activity rescued by a heterozygous LMNA null mutation causes a severe, HGPS-like, MADB phenotype, whereas partial accumulation of prelamin A with reduced ZMPSTE24 enzyme activity is associated to milder MADB phenotypes The patient reported here during the 30 year follow up developed a MADB phenotype associated with a congenital myopathy, further extending the spectrum of clinical features associated to ZMPSTE24 mutations. Furthermore, our patient's MADB phenotype seems to recapitulate, to some extent, the main features of RD. Intrauterine growth retardation, tight skin and arthrogryposis at birth are classical features of RD. The disease evolved progressively and the whole clinical phenotype such as failure to thrive, mandible and clavicle hypoplasia, distal acro-osteolysis, scleroderma like skin with subcutaneous calcifications appeared during the 1 st and 2 nd decades followed by vascular complications including high blood pressure and atherosclerosis as observed in other MADB patients. To our knowledge, this is the first report describing congenital myopathy in a patient affected with MADB.

The relatively mild progeroid phenotype of our patient is well correlated with the western blot analyses which showed highly reduced expression of ZMPSTE24, with residual activity, since reduced levels of mature lamin A and partial prelamin A accumulation coexist in the patient's cells. In addition, according to the in silico studies which predicted two conformational states of the mutated ZMPSTE24, we can imagine that one part of the mutated ZMPSTE24 is correctly folded despite the presence of mutation and is still functional, allowing normal maturation of prelamin A, whereas the other part missing the second TM domain and sharing enzymatic site and ER signal in the ER lumen, is either degraded or unable to cleave prelamin A. The existence of both conformations could explain both the reduced level of ZMPSTE24 and the partial prelamin A maturation detected by western blot.

At the cellular level, the coexistence of both prelamin A and lamins A and C leads to reduced life span of the cells with major nuclear abnormalities. [START_REF] Navarro | Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganisation and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Goldman | Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome[END_REF] From a phenotypic point of view, our patient suffered from particular features of prognostic value including impressive subcutaneous and vascular calcifications. The unusual paravertabral diffuse calcinosis led to spinal cord compression requiring surgery. Such calcifications have been reported in 3 ZMPSTE24 related MADB cases, 2 of them requiring surgical removal, [START_REF] Agarwal | Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia[END_REF]36 but not in the 2 others. [START_REF] Miyoshi | Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings[END_REF] Similarly, a homozygous LMNA mutation c.1718C>T leading to the missense substitution p.Ser573Leu has been reported to result in progeroid features associated with arthropathy and tendinous calcinosis [START_REF] Van Esch | A homozygous mutation in the lamin A/C gene associated with a novel syndrome of arthropathy, tendinous calcinosis, and progeroid features[END_REF] without any prelamin A accumulation. Also, renal artery calcifications and chronic kidney disease were responsible for pharmacoresistant high blood pressure as observed in ZMPSTE24 related MADB. [START_REF] Agarwal | Focal segmental glomerulosclerosis in patients with mandibuloacral dysplasia owing to ZMPSTE24 deficiency[END_REF] The glomerular and tubular abnormalities observed in our patient argue for a multifactorial aetiology.

In addition to progeroid features, our patient developed a clear and proven myopathy. She showed evidence of skeletal muscle involvement including muscle weakness with scapular winging, myopathic EMG pattern and fibre type disproportion with a predominance of small type I fibres and early respiratory insufficiency. These finding were unusual in the context of a ZMPSTE24 related progeroid syndrome. Skeletal muscle abnormalities, including muscle atrophy, first proximally (scapulae, buttocks) then distally in the limbs, are classical symptoms described in HGPS. [START_REF] Hennekam | Hutchinson-Gilford progeria syndrome: review of the phenotype[END_REF] As the diminished joint mobility observed in HGPS patients does not appear to be due to tightening of the skin, one may hypothesize that it may be due to muscle sclerosis and tightness similarly to what is observed in myopathies. The emergence of LMNA gene mutations in HGPS patients prompted the examination of skeletal muscle leading to the description of the first cases with progeroid syndrome and confirmed early onset myopathy due to p.Ser143Phe LMNA substitution [START_REF] Kirschner | S143F mutation in lamin A/C: a new phenotype combining myopathy and progeria[END_REF][START_REF] Madej-Pilarczyk | Progeria caused by a rare LMNA mutation p.S143F associated with mild myopathy and atrial fibrillation[END_REF] or type A MAD due to p.Arg471Cys homozygous [START_REF] Zirn | Association of homozygous LMNA mutation R471C with new phenotype: mandibuloacral dysplasia, progeria, and rigid spine muscular dystrophy[END_REF] or p.Arg527His / Val440Met compound heterozygous [START_REF] Lombardi | Compound heterozygosity for mutations in LMNA in a patient with a myopathic and lipodystrophic mandibuloacral dysplasia type A phenotype[END_REF] LMNA mutation. Among the 30 previously reported ZMPSTE24 mutated patients; none had obvious or proven skeletal muscle involvement. While in our patient the first symptoms linked to muscle involvement appeared at 2.5y, RD patients never reach this age and usually die in the first hours/days after birth. Interestingly, even if none of the 5 ZMPSTE24 related MADB cases 32- skeletal muscle fatty infiltration in gluteal and thigh muscles similar to what is described in other muscle diseases. On the other hand, Zmpste24 knockout mice [START_REF] Pendas | Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice[END_REF][START_REF] Bergo | Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect[END_REF] are normal at birth but show slightly variable phenotypes. Skeletal muscle involvement manifested mainly by abnormal gait and muscle weakness with either a normal histological aspect [START_REF] Bergo | Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect[END_REF] or a significant increase in abnormal small round fibers. [START_REF] Pendas | Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice[END_REF] The myopathic signs in our patient were similar to those observed in Zmpste24 knockout mice. The etiopathogenic mechanism of skeletal muscle involvement in our patient remains to be determined. Unfortunately, muscle cells of the patient could not be explored but, even if we cannot rule out the possibility that muscle disease is linked to one or more modifier genes, we suppose that they may exhibit reduced lifespan due to prelamin A accumulation, as do fibroblasts. Further skeletal muscle explorations in patients affected with MADB due to ZMPSTE24 loss of function might help to clarify this issue.

Titles and legends to figures

  1 ), and left clavicle (figure 1,b 1-4 ) were detected by 5mo. Enlarged anterior fontanel, delayed skull ossification and tooth eruption (figure 1, c 2 ) were noted at 9mo, but after 4y teeth rapidly crowded (figure 1, c 3 ). Other bone abnormalities included femoral head dysplasia, and osteolysis, delayed bone maturation and distal finger osteolysis (figure 1, c 5-6 ). Long bones had characteristic enlarged metaphysis and slender diaphysis (figure 1, c 5 ). Decreased spine and limb bone density was associated with multiple spontaneous fractures, first observed in a clavicle at 6y (figure 1, b 5-6 ), and later in ribs, vertebrae and femur (figure 1, b 7 ). At 13y, osteolysis of the 3 rd and 4 th cervical vertebral spine process accompanied by C3-C4 and C7-D1 anterolisthesis occurred (figure 1, b 8 ). Cranial sutures were still open at 15y (figure 1, c 7 ).

Figure 1 .

 1 Figure 1. Patient clinical features. a. Photographs of the patient at different ages showing

Figure 2 .

 2 Figure 2. Characterisation of the ZMPSTE24 mutation. a. Genomic DNA sequences of

showed such myopathic involvement, one of these cases (patient 1 in[START_REF] Miyoshi | Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings[END_REF] ) showed clear

ACKNOWLEDGMENTS

We wish to thank Jean-Paul Leroy for his help in the clinical investigation of the patient, Thomas Voit for critical reading and fruitful discussion and the Myocastor study group for its constant support. This work was financially supported by the Institut National de la Santé et de la Recherche Médicale (INSERM); the Université Pierre et Marie Curie Paris 06 (UPMC); the Université de la Méditerranée, the Centre National de la Recherche Scientifique (CNRS); the Association Française contre les Myopathies (#11057, #11034); the European Union Sixth (Euro-laminopathies #018690) and Seventh (Myoage #223576) Framework Programmes.

Conflict of Interest Statement:

The authors declare no conflict of interest.

ZMPSTE24 in patient's cultured skin fibroblasts is associated with accumulation of prelamin A.


Olivier Valentin

Eric Blond

Nicolas Richet

Towards a complete macroscopic modelling of the thermo-chemomechanical behaviour of MIECs

Keywords: MIECs, Chemical expansion, Thermochemo-mechanical behaviour

This paper proposes a macroscopic model of the thermo-chemo-mechanical behaviour of MIECs dedicated to mechanical engineering. Developed within the framework of phenomenological approach, the state variables of the model are the oxygen partial pressure, the temperature and the total strain. Oxygen bulk diffusion is describes by the local Onsager approach and surface exchange are modelled by the use of a mass Biot number. Not only focus on the semi-permeation induced strain, the proposed model also included the effect of the temperature. Strain predicted by a model based on defect equilibrium and strain predicted by the macroscopic approach are in good agreement as it is illustrates on the particular case of La 0.8 Sr 0.2 Fe 0.7 Ga 0.3 O 3-δ . The implementation of the model in the F.E.A code Abaqus allows the exploration of the evolution of the stress state during the membrane life, from sealing stage to reactor exploitation.

Introduction

Since more than twenty years, MIECs are promising sources of hot pure oxygen which have potential industrial applications like methane reforming or oxycombustion. Two fundamental keys points for the development of these technologies are the lifespan and the reliability of the membrane. The first point is mainly driven by the stability of the material under operating condition whereas the second point is directly linked to the mechanical stress induces in service 1 . A lot of works focus on the material properties which ensure a high permeation rate and a good stability but few study deals with the mechanical reliability under operating conditions. The development of a model dedicated to the field of mechanical engineering for MIECs materials is necessary to investigate the reliability in service.

The work presented hereafter focus on the development of a macroscopic model of the thermo-chemo-mechanical behaviour of MIECs accounting for oxygen permeation, thermal expansion and strain induced by the stoichiometry fluctuation. While the previous works have been firstly focus on the semi-permeation induced strain 2,3 , the model in its current form take into account the effect of the temperature on the chemical expansion due to the defect equilibrium dependence to temperature. To test the ability of this model to predict the chemical strain, its results are compared to those of a more classical model based on the defect equilibrium considering the particular case of La 0.8 Sr 0.2 Fe 0.7 Ga 0.3 O 3-δ (LSFG) for which the chemical strain under atmosphere have been extensively studied 4,5,6,[START_REF] Valentin | Solid States Ionics in press[END_REF] .

Finally, the current numerical implementation of the model in the F.E.A code Abaqus allows to investigate the stress state of a tubular membrane for syngas production taking into account the residual stress induced by each stages, from sintering to in-service sollicitations via sealing.

Thermomechanical behaviour

The chemical expansion is related to cations size variations or/and to the modification of the Coulomb repulsion between the defects induced by oxidation and reduction of the material. Various approaches to describe this phenomenon exist in the literature [START_REF] Kharton | [END_REF][9][10][11][12][13][14] . The model proposed hereafter is an extension of that presented in ref 2,3,15 .

Chemical induced strain does not modify the elastic mechanical behaviour. Assuming the partition of small strains leads to write: (1) Where is the strain second rank tensor divided in three parts: the thermal strain, the elastic strain and the chemical strain. In the rest of this paper, bold type refers to tensor, while normal type refers to scalar. Then, the stress -strain relationship is: :

Where K is the four rank Hooke tensor and the second rank stress tensor. The thermal strain tensor depends on temperature variation in a reversible way:

Proceedings of the 12 th Conference of the European Ceramic Society -ECerS XII Stockholm, Sweden -2011 2(4)

Where α is the coefficient of thermal expansion, T 0 a reference temperature and I the second rank identity tensor. The thermal deformation corresponds here exclusively to the deformation caused by temperature variation at constant composition and without any phase change.

Chemical expansion model

The chemical strain is due to the oxydation state of the material. For a constant temperature, the oxydation state is drive by the oxygen partial pressure of the atmosphere, whereas at constant oxygen partial pressure the oxydation state is drive by the temperature. Indeed, the Gibbs law, and so the temperature, drive the stoichiometry of the defect equilibrium. But, the equilibrium with the surounding atmosphere impose the oxygen activity in the material, and so the defect stoichiometry too. Then, a macroscopic model of the chemical expansion behaviour should take into account the effect of temperature and oxygen partial pressure. Moreover, assuming an isotropic chemical strain, only the scalar expression which multiplies the second rank identity tensor needs to be defined.

The first assumption of the proposed approach is the reversible dependence of the chemical strain with oxygen partial pressure variation. This assumption has been previously validated by isothermal expansion and XRD measurement under different atmosphere on different MIECs [START_REF] Valentin | Modélisation thermo-chimiomécanique des conducteurs mixtes Application à la production de H2/CO[END_REF] . The second assumption is that, thanks to the admissible stoichimetry fluctuation in accordance to Gibbs rule, the temperature drives the range of the chemical expansion induced by the atmosphere change. Then, it leads to:

ln (4)
The first term of (4) corresponds to the part of the chemical expansion induced by the atmosphere change, in agreement with the previously proposed expression 15 . In this term, β is the chemical expansion coefficient, P O2 the current oxygen partial pressure and P ref the oxygen partial pressure corresponding to chemical strain equal to zero (i.e. often equal to oxygen partial pressure of the sintering atmosphere). The second part, , represent the chemical strain induce by the temperature change. The latest term, , is the maximum chemical strain amplitude which corresponds to the larger acceptable stoichiometry fluctuation for the considered material.

In this step towards a complete macroscopic model, let us proposed a linear expression for the thermal effect on chemical expansion: (5) Where α c is the thermal chemical expansion coefficient, is the temperature of activation of this effect and is the positive part of x. The identification of , , , requires cyclic thermal expansion test with atmosphere change between each cycles [START_REF] Valentin | Modélisation thermo-chimiomécanique des conducteurs mixtes Application à la production de H2/CO[END_REF] . For LSFG, the obtained values are 2.5 10 , 2. 10 , 500° , 0.25%. Then, it is possible to plot the evolution of chemical strain versus oxygen partial pressure and temperature, like proposed on figure 1. Results predicted by this macrocscopic model can be compared to those of a model based on the defect equilibrium and identified for LSFG [START_REF] Valentin | Solid States Ionics in press[END_REF] . Figure 7 presents prediction of this second model, assuming that the equilibrium defect is never reach under 700°C, then, the strain is "frozen" for lower temperature. It is worth nothing to underline that, without this assumption, a model based on the defect equilibrium will always predict no strain at room temperature because it coresponds to the thermodynamic equilibrium, that will be never reach. Noting that the origin of strain is different on figure 1 and 2, it can be observed that the two predictions are in agreement. The average gap between the models is around 6% and locally it never exceeds 14 %. The defect based model better fit with strain estimate by XRD measurement of lattice parameter whereas the macroscopic model better fit with thermal expansion measurement. For the two models, the gap between associated experimental values does not exceed 5 %. The main differences are: the activation temperature; the total amplitude; the shape of the strain step. While the defect model considers solely the stoichiometry change in respect to defect equilibrium, the macroscopic approach encompasses all phenomena that can induce a stoichiometry change (e.g. diffusion). Indeed, for its identification, the macroscopic approach does not need to assume the equilibrium of defect but only the equilibrium of the oxygen activity and temperature between the material and the surrounding atmosphere. It may explain the difference in activation temperature. The maximal chemical strain is higher from the macroscopic measurement (i.e. thermal expansion) than that extrapolate from XRD measurement of the lattice parameter change. Same little scattering are observed for the thermal expansion, and it may be justified by the scale effect. The shape of the strain step predicted by the defect model is smoother than that of the macroscopic model because of the assumption of a linear thermal dependence. Obviously, this thermal dependence should be linked to the evolution of the equilibrium constant, and it is not truly linear. This point will be better treated in a close future.

Semi-permeation model

The oxygen bulk diffusion can be model using the Onsager principle, which leads to the local expression of the bulk density of oxygen flux:

⁄ ln ( 6
)
Where a is the oxygen activity, S 0 the "intrinsic" oxygen diffusivity, Q the activation energy and R the ideal gas constant. For LSFG, value of S 0 =100 m 2 .s -1 and Q=138 kJ have been estimated by inverse identification [START_REF] Valentin | 12th ECerS[END_REF] .

The surface exchange steps correspond to the adsorption and dissociation of the reacting gas molecule and the incorporation of the dissociated species. Considering equilibrium at surface between the oxidation and reduction rate, the oxygen surface flux is described by the equation [START_REF] Bouwmeester | [END_REF] :

ln ( 7
)
Where k is the surface exchange coefficient. While in steady state the knowledge of the limited step is enough to determine the oxygen activity gradient in the membrane, in transient stage this gradient change with time and space depending on the ratio between surface and bulk kinetics. The dimensionless Biot number B i can be used to characterized the bulk to surface exchange ratio:

Where e is the thickness of the membrane and D the oxygen diffusivity at the considered temperature. A high Biot correspond to a bulk limited permeation while a low Biot corespond to a surface limited permeation. A Biot in the range around 0.6 to 10 coresponds to mixed regime 15 .

Numerical simulations

The mechanical behaviour accounting for the chermical strain (4) has been implemented in the finite element code Abaqus thanks to the user subroutine UMAT. The bulk diffusion behaviour (7) has been also implemented in Abaqus using the apropriate user subroutine UMATHT. The surface exchange flux (7) has been implemented thanks to the user subroutine DFLUX. Figure 3 and4 show the comparison between experimental results of thermal expansion test under atmosphere and numerical prevision. The strain amplitude is quite good whereas the kinetic does not fit the curves. Kinetics seems too fast under air and too slow under nitrogen. It underlines that the simplified semi-permeation model used here need to be refined. However, considering only the case of stationary states, the chemical strain is quantitatively good. Then, it allows to use the model to estimate the mechanical stress induce by stoichiometry change in stationary state. To illustrate the possibility offer by such macroscopic model, the case of a tubular membrane of LSFG, close at one end, has been study. After sintering, the membrane is sealed at its based. This process generates mechanical stress that can damage the tube. Then, the membrane is set in the methane reforming reactor to produce syngas. Feed air arrives inside, while methane flow is at the outside face. Before reach the steady production parameters in term of atmosphere and temperature, different intermediate state need to be passed. Then, after a reasonable production time, the reactor may need to be stopped. Each one of this stage may impact the stress field reach in the next stage and could damage the membrane. Then, all the solicitations have to be computed accounting for the cycle process. Figure 5 shows the evolution of the maximal principal stress at different localization in the membrane. The red line describes stress evolution near the sealment, the green one near the closure on the top and other colors corespond to intermediate level. Firstly, it is assumed that the membrane is stress free at the beginning of the cooling of the sealment under air. So, after cooling, the thermal expansion coefficient mismatch between LSFG, sealment material and steel, combine to the geometry of the sealment, produce a high stress close to the tensile strength. After being installed in the reactor, the start up process can begin. The heating up under neutral gas permit to relax stresses while switching to air on inner face and methane on outer face induce an increase of the mechanical solicitations, but stresses still being lower than after the sealing. Finally, for the example, the particular case of an inappropriate gas atmosphere management during the stop of the reactor is simulated and it is observed that it can produce the fracture of the membrane.

Conclusions

The macroscopic model briefly presented in this paper has been developed to study the effect of reactor design or management on mechanical reliability of the membrane. The temperature and the partial pressure have been deliberately choose as state variables because they can be "easily" measure and manage in service. The results show that, whereas the chemical expansion is well reproducing, the surface exchange needs to be refined. However, the model permit to quantify the mechanical stress in the membrane in steady-state service condition, accounting for geometry and mechanical design of each part of the reactor. Such macroscopic model dedicated to mechanical engineering of MIECs should give a help to the development of industrial application of these promising materials.

Figure 1 :

 1 Figure 1: Macroscopic model of the chemical strain

Figure 2 :

 2 Figure 2: Microscopic model of the chemical strain

  nitrogen / 900 °C (5) air / 400 °C (6) nitrogen / 400 °C (1) air / 900 °C (2) air / 1100 °C (3) nitrogen / 1100 °C Proceedings of the 12 th Conference of the European Ceramic Society -ECerS XII Stockholm,

Figure 3 :

 3 Figure 3: Thermal expansion under nitrogen for LSFG sintering under air.

Figure 4 :

 4 Figure 4: Thermal expansion under air for LSFG initially stabilized under nitrogen (starting point at 0.2% of strain at RT).

Figure 5 :

 5 Figure 5: Evolution of dimensionless maximum principal stress at different localization along the membrane life.