Maria-Susana Seguin

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Introduction :

It seems difficult, in the context of a reflection on the radical thought in the early moderns period, not to address the role played by clandestine philosophical literature in the elaboration and circulation of the most controversial ideas. The researches carried out during the last thirty years have definitely proved the importance of this underground movement which has even modified the traditional historiography of the Enlightenment. It seems difficult also, thinking on the place of women in radical thought, not to mention the name of Émilie Du Châtelet, symbol of the woman philosopher ("femme philosophe") of the French Enlightenment. The Marquise has not only translated Newton or Mandeville, she realized her own scientific experimentations, debated with philosophers and scientists of her time and thus defied the dominant masculine thought. 1 But Émilie Du Châtelet goes even further: she is also the author of one of the most virulent clandestine philosophical manuscripts against religion : the Examens de la Bible 2 . But may we recognize in Madame Du Châtelet's work the particular mark of a feminine thought, different from that of the other authors of this complex corpus ?

In order to analyze this question, I would like to articulate my reflection in three steps : First, a brief review of the main characteristics of the clandestine philosophical literature into which the Examens de la Bible by Madame Du Châtelet are included ; secondly, the elements that, in Émile Du Châtelet's work, can appear as characteristics of a feminine writing ; finally, a quick comparison of the Examens with other works in the clandestine corpus attacking the biblical writings.

The clandestine philosophical literature:

From the perspective of historical research 3 , the corpus of clandestine philosophical literature is composed of manuscript texts which have, since they were first identified by 1 Véronique Le Ru, Émilie Du Châtelet philosophe, Paris, Classiques Garnier, 2019. 2 Gabrielle-Émilie Le Tonnelier Du Breteuil, Marquise Du Châtelet-Lomond, Examens de la Bible. Édités et annotés par Bertram Eugene Schwarzbach, Paris, Editions Honoré Champion, collection « Libre pensée et Littérature Clandestine », 2011. 3 On this subject, see Geneviève Artigas-Menant : "Cent ans de réponses aux 'Questions diverses' de Lansonˮ, Problemata. Revue Internationale de philosophie, Vol. 4, 3, 2013, pp.21-49. Maria Susana Seguin, "Quelques réflexions sur les auteurs de Gustave Lanson 4 and Ira O. Wade 5 continued to expand, according to the meticulous investigation conducted by Miguel Benitez and completed by international collaborations 6 , to the point of encompassing 335 different titles in more than 2500 copies, held mostly in European, and in particular French, libraries. A covert circulation of all this texts is proved by eyewitness accounts, correspondence, the presence of these titles in the catalogues of public or private libraries, or in inventories made after death, etc. The coherence of this corpus does not, however, meet an observable material criterion, much less amount to a declared desire on the part of the authors composing it to constitute a distinctive intellectual movement or to stoke a philosophical debate -at least, not in every case. This corpus has been constituted as such a posteriori by the researchers themselves, who have detected very strong elements of commonality in these scattered writings, who have identified their channels of production, circulation and reception, and demonstrated that, more than being the result of a specific, concerted intention, this corpus is symptomatic of a transformation whose significance was sometimes lost on the authors themselves 7 .

Which criteria may be used to define this corpus ? As far as its nature is concerned, it is composed essentially of manuscript copies, as was until recently the case for the texts of Madame Du Châtelet, distributed without the name of the author, or with sibylline attributions, whose dissemination did not necessarily precede a final printing stage. There are copies based on printed works that are now rare (such as, for example, the Cymbalum mundi by Bonaventure des Périers 8 ), manuscripts that were in fact printed subsequently, in the great majority of cases, by someone other than the author (as in the case of the Doutes sur la 6 Reference should be made here to the very important inventorial work carried out by Miguel Benítez in La Face cachée des Lumières, Paris: Universitas, Oxford: The Voltaire Foundation, 1996, and which will be further developed in the Spanish edition of the same work, La Cara oculta de la Luces, Valencia, Biblioteca valenciana, Ideas series, 2003. La Lettre clandestine regularly announces the discovery of new copies of previously inventoried texts or the existence of new titles that may be entered into the clandestine corpus. The entirety of the corpus of clandestine philosophical literature can now be consulted at the digital platform philosophie-clandestine.huma-num.fr, which is regularly updated, to which we will refer from now on. 7 See the various thematic issues of La Lettre clandestine devoted to the problems associated with the appellation and description of the clandestine corpus, such as "Tendances actuelles dans la recherche sur les clandestins à l'âge classiqueˮ (5 / 1996), "L'identification du texte clandestin aux XVII e et XVIII e sièclesˮ (7 / 1998), or "Anonymat et clandestinité aux XVII e et XVIII e sièclesˮ (8 / 1999). On the relationship between clandestine literature and the intellectual life of the period, in addition to the issues devoted to the major authors of the Enlightenment, see also "Protestants, protestantisme et pensée clandestineˮ (13 / 2004), "Les matérialismes dans la littérature clandestine de l'âge classiqueˮ (14 / 2005-2006), "Les Relations franco-anglaises aux XVII e et XVIII e siècles: périodiques et manuscrits clandestinsˮ (15 / 2007), "La littérature philosophique clandestine et les sciencesˮ (18 / 2010). 8 http://philosophie-clandestine.huma-num.fr/ms/34. religion, attributed to Dumarsais and published by Voltaire in his Évangile de la raison 9 ), often long after they had died (as with the Mémoires des sentiments et des pensées de Jean Meslier 10 ). There are, evidently, manuscripts in existence that would have been impossible to distribute in printed form within the context of the censorship of the first half of the eighteenth-century. These include the famous Traité des trois imposteurs, whose composition may perhaps be dated back to the last years of the seventeenth-century but which was not printed, through the efforts of Baron d'Holbach, until 1768, while the circulation of the manuscript version of the text throughout the eighteenth-century made it the most widely diffused clandestine philosophical text and the manuscript version even continued to be diffused at the same time as the printed version 11 . There are, however, numerous manuscripts that have remained anonymous and unpublished until the present day, or which have only been published as a result of contemporary research, such as, in particular, the manuscript that interests us here, the Examens de la Bible by Madame Du Châtelet 12 .

The material criterion (the manuscript form) is not, however, sufficient to situate this text within the corpus with which we are concerned. The intellectual and material history of reading and the book tells us that the various forms assumed by clandestine philosophy (manuscripts, publication outside France, secret publication within France, etc.) constitute one of the specific configurations of intellectual life of the eighteenth-century, that adopted by a substantial number of works which, for various reasons, evade the strict requirements imposed by the publishing market and the implementation of royal and religious censorship, in other words, the majority of the texts 13from controversial works of every type to religious literature identified as heterodox (Protestant or Jansenist), pornographic literature and, even, the simplest novel, at least up to the middle of the century 14 .

The texts of clandestine philosophical literature are therefore characterised by the fact that in addition to their covert diffusion, anonymity or false attribution, and their manuscript form, they are "philosophicalˮ in the broader sense attributed to this term by the Enlightenment, that defined precisely by the article "Philosopheˮ in the Encyclopédie, and which was extracted from one of the clandestine philosophical manuscripts15 . They deal in a critical, subversive or 9 http://philosophie-clandestine.huma-num.fr/ms/36. 10 http://philosophie-clandestine.huma-num.fr/ms/122. 11 http://philosophie-clandestine.huma-num.fr/ms/189. See the issue devoted to this text by La Lettre clandestine 24 / 2016. 12 http://philosophie-clandestine.huma-num.fr/ms/28. 13 On this subject see H.-J. Martin and R. Chartier, eds., Histoire de l'édition française, vol. II, Paris, Promodis, 1984. 14 Françoise Weil L'Interdiction du roman et la librairie: 1728-1750. Paris: Aux Amateurs de livres, 1986. See also Livres interdits, livres persécutés: 1720-1770. Oxford: Voltaire Foundation, 1999. impious spirit with essentially metaphysical or religious questions while combatting prejudice through rational reflection (philosophical, historical and scientific reason). These are, thus, for the most part heterodox writings, to the extent that in the majority of cases the ideas that they condemn concern the tenets of the faith: God himself (his existence, his essence and his relationship with the world); the human soul (its spirituality and immortality); revelation (authenticity or coherence of the Biblical scriptures, validity of the prophecies and miracles, strength of exegesis); the historical religions in general (their origins, their relationship with the political world, the crimes committed in their name in the course of history), and Christianity in particular, especially Catholicism, through its history, dogmata, mysteries and morality. However, this constant factor within the field and the critical process may rest upon highly varied philosophical orientations, from naturalism to materialism, from atheism to pantheism or deism, and reflect the influence of Descartes, Bayle, Hobbes or Spinoza, and the libertins érudits of the seventeenth-century.

The Examens de la Bible by Madame Du Châtelet fall within the definition of the clandestine corpus. Without entering into a detailed analysis here, as Bertram Schwarzbach has done in a highly apposite manner in his introduction to the edition of the text, we are able to state that the work produced by Émilie satisifies these criteria, both in terms of its material character (an anonymous manuscript work, at least until recently), and its methodological and philosophical approach. Thus, the treatise presents a critical commentary of each of the books of the Bible, subverting the apologetical method of the period, in particular that of the model of the genre, Augustin Calmet, who at the beginning of the century had authored a Commentaire de la Bible which Voltaire would later adopt as one of his preferred targets 16 .

And finally, through the systematicity of the selected method, Émilie Du Châtelet puts the Christian religion, and Catholicism in particular, on trial by attacking the very foundations of Christian theology (the scriptures), employing all of the angles of attack characteristic of clandestine philosophical literature: denunciation of revelation 17 , absurdity of the prophecies 18 and miracles 19 , immorality of the Old Testament 20 , contradictions in the Gospel texts 21 , etc. 16 Dom Augustin Calmet, Commentaire littéraire sur tous les livres de l'Ancien et du Nouveau Testament, Paris, publ. Pierre Emery, 1707-1717, 26 volumes. 17 By way of example, Du Châtelet writes: "Que l'on compare ce passage avec le Lévitique et qu'on voye si Dieu a pu inspirer l'auteur du Lévitique et les prophètes qui lui donne[nt] si positivement le démentiˮ, "Examen du Lévitiqueˮ, Examens de la Bible, ed. cit., p.229. 18 There are numerous examples of this type of criticism. Consider, for instance, the commentary authored by Du Châtelet concerning a prophecy of the Old Testament: "Le reste de la prophétie de Jacob est dans ce goût là et ressemble aux discours d'un homme qui a un violent transport au cerveau et dont les discours n'ont ni sens ni têteˮ (ibid., p.195). 19 Once again, there are numerous examples of this. We may consider one example that summarises the position adopted by the Marquise: "Voilà donc l'histoire du Pentateuque finie, il faut avoüer que c'était bien la peine de faire tant de miracles et tant de mal aux hommes pour faire sortir les Israelites d'un beau et bon pays comme l'Égypte […]ˮ. ibid., p.280.

The manuscript thus subjects the Bible, book after book, to a rational reading whose arguments are derived from historical and scientific reasoning, the critical use of apologetical sources, as well as an internal analysis of the work for the purposes of demonstrating its human nature, its imperfections and therefore the impossibility of it conveying a particular revealed message and imposing any kind of universal morality. Through its choice of methodology (attacking the founding texts of the Christian religion) and the strength of the criticism offered, the Examens of Madame Du Châtelet constitute one of the most systematic and effective clandestine philosophical manuscripts of the first half of the eighteenth-century, and appear to complete the intellectual output of this exceptional woman by bringing a new dimension to her already rich and influential work. This would also help to explain the cloak of anonymity under which this manuscript has reached us, as well as the profane legend that has developed over time concerning the author and her philosophical intentions.

The Examens de la Bible, a feminine philosphy ?

These elements does lead us to examine which allows (or not) to define this manuscript as particularly "feminine", or, more, as "feminist". We must not forget that the clandestine work of Madame du Châtelet has reached us anonymously and has been attributed to her with certainty only after several years of a very precise research by the modern editor of the text, Bertram Schwarzbach (who had nevertheless refused to attribute this manuscript to Madame Du Châtelet for years). Certainly, a form of legend ran from the last years of the eighteenthcentury, which attributed to Émilie "a commentary of ten volumes on the Bible." This is essentially what can be read in a correspondence between Condorcet and Turgot, in 1772, which did not attribute this text to Émilie alone, but to the collaboration of the Marquise, Voltaire and even Saint-Lambert, Emilie's last lover. 22 Doubt therefore remained about the reality of this attribution as well as about the role that Voltaire could actually play in the composition of this manuscript.

One of the elements, and not least, analyzed by Bertram Schwarzbach in his edition will have been "the feminine part" or even "the feminist one" of the Examens de la Bible, which could indicate that the author of the manuscript is a woman. I will not return here to this work, 20 "Mais elle [la bru de Judas] envoya à Judas avant d'aller au bucher le gage qu'elle avait reçu de luy; ce qui luy sauva la vie quoique par là, elle eut joint l'inceste à l'adultèreˮ. ibid., p.189. 21 Hence, regarding the genealogy of Jesus she writes: "Or Joseph n'etant que le pere putatif de Jesus, et n'y ayant rien mis du sien, autant valoit-il faire la généalogie de Mahomet que la sienne pour donner celle de J[ésus] C[hrist]. Mais cette généalogie si absurde à raporter, fourmille de contradictionsˮ. ibid., p.614. 22 Letter from Condorcet to Turgot, 14 June 1772, letter LXVII, in Correspondance inédite de Condorcet et de Turgot 1770-1779, éd. Charles Henry, Paris, 1883 ; Genève, Slatkine reprints, 1970, p. 87. the critical edition of the manuscript does so very methodically in the very long introduction to the text. In fact, the attribution of the Examens de la Bible to Émilie Du Châtelet can no longer be challenged. However, I retain here some points necessary to complete this analysis. Some textual clues, but very weak, seem to indicate a woman author: on one of these three preserved copies of this manuscript, we note the presence of five adjectives or feminine verbal forms of the past (in French) and which seem to indicate a female speaker [START_REF]Examens de la Bible[END_REF] . But these elements also seem too insignificant, in the context of a very large manuscript, to constitute a definitive proof: none of the three known manuscripts of the Examens de la Bible was written by the hand of Emilie, for example. These cases may also be the consequence of a simple transcription mistake especially when we know the relative rigor of the work of the copyists of the time.

The interest that the author of the manuscript gives to women of the Bible appears, however, as more interesting for us: Madame Du Châtelet is particularly interested in the place that the Bible gives to women, often subject to male interests. She criticizes Abraham, who twice wanted to prostitute his wife Sara 24 , or who put his concubine Hagar out, left to herself in the desert, because of the jealousy of his wife 25 . She admires Sephora, Moses' wife, who had the courage to circumcise his own son to save him from the angel who wanted to kill him 26 . Or the strength of Abigail who protected his first husband, Nabal, who had refused to help King David, the one who will later become her husband 27 .

The author of the manuscript also demonstrates a sure knowledge of the feminine and family life. She attaches importance, for example, to Joseph's place in the education of the young Jesus 28 and to the delicacy that some women can show in the relationship with their husbands. She is also interested in the intimate life of women, as well as their sexuality: she offers explanations on the nature of menstrual blood 29 and discusses the conventional wisdom regarding the question of women's purity after childbirth (especially after the birth of a girl) 30 . She uses this knowledge above all to mock religion, as in the case of one Capuchin who claimed to be able to take on the pain caused by childbirth 31 . From the moral point of view, she takes the defense of adulterous women, especially that mentioned in chapter 8 of the book of John, unfairly accused by society and especially by men, whose she denounces the recurring infidelities 32 . On this point, she does not even spare the figure of Jesus: his proximity to several "holy women" during his travels seems to her very suspicious, and the nature of his relationship with Mary Magdalene does not seem to make any doubt, even if she's not shocked by this relationship 33 . Émilie also makes fun of the need for purity that weighs on young women and mentions that there are many ways to simulate lost virginity 34 .

The author of the Examens, however, is not always supportive of female turpitude : she condemns Esther, who agreed to spend a night with Ahasuerus before deciding on her future 35 , and condemns Ruth, who slept with the wealthy Boaz to force him to marry her, because she has done nothing but prostitute herself 36 . But these critics are not made in the name of Christian morality: the case of Judith story offers a very interesting counterexample, because she is the model of a stupid prudery, which does not prevent her from being also very bloodthirsty 37 .

Can we deduce from these arguments that the text brings a feminine, even a feminist vision of Biblical criticism? The question is not easy, because Madame Du Chatelet's Examens de la Bible are the only clandestine philosophical manuscript written by a woman that we have been able to identify to this day. We can only compare it to the critical practices of other manuscripts of the same period, which approach the biblical writings with the same polemical intent.

The Exmens in the context of clandestinity:

On this point, it is obvious that the text of Madame Du Châtelet appears as original. A manuscript like L'Analyse de la religion chrétienne, anonymous, also proceeds to the systematic commentary of some Biblical passages but does not seem to give the same place to the problematic of the woman. Admittedly, the intention of the text is not, as in the case of Madame Du Châtelet, to read in a precise way each chapter of the Holy Scriptures, and the author skips many passages that the method chosen by Émilie cannot ignore. But the author of the Analyse is not interested in the matriarchs of the Old Testament, nor in the sexual life of women, except to point out the moral contradictions inherent in a supposed revealed book. 32 Ibid., p. 781. 33 Ibid., p. 747, 801-802. 34 Ibid., p. 275. 35 Ibid., p. 488-489. 36 Ibid., p. 325. 37 Ibid., p. 478-483.

Another clandestine manuscript contemporary to that of Emilie, whose author might have known of the Examens de la Bible, according to Bertram Schwarzbach, can also serve here as a piece of comparison. It is a text conceived as a complement of the preceding one, and which bears the title of Preuves citées dans la Religion chrétienen annalysées sans y être rapportées 38 . In this text, the anonymous author offers a systematic reading of all the books of the Old Testament and thus crosses the same stories that Émilie comments. However, again, he does not dwell on the cases evoked by the Examens, or if he does, it is not according to the same method. If Sarah's case is of interest to this author, it is to denounce the absurdity of her motherhood at more than 90 years old. But the behavior of Abigail, Esther or Ruth elicits no comment, except doubts about the author of the books that relate their stories. Only one case deserves our attention, however, it is the story of the rape of Dinah, reported by chapter 29 of Genesis, which could have interested Emilie but she does not comment. The anonymous author of Les Preuves, who builds his criticism of the Bible on scholarly chronological calculations and historical comparisons, is astonished less by the violence perpetrated on a 7year-old girl by an adult, than by the improbability of the rest of the story: the many children that Jacob was able to father at the age of 85 and especially the age of Simon and Levi, Dinah's brothers who kill all the men of the city of Salem to avenge the honor of their sister, when they are only 11 and 12 years old ... 39 An obvious thematic difference emerges when we compare Émilie Du Châtelet's Examens with other clandestine manuscripts of the same period. The choice of systematicity in the criticism of the Bible could, it is true, explain Emilie's attention to feminine stories, despite some omissions. But, on the other hand, this seems to correspond more to a real interest in the status of women in Holly scriptures and in religion and in the consequences that these readings may have in Émilie's time. The difference is also methodological: Émilie Du Châtelet does not appeal to historical or chronological arguments, like the author of the Preuves, whose criticism is much more scholarly. But basically, the intention of both authors remains the same: to denounce the absurdity of the Bible and the morality that follows. To denounce, on the name of reason, and not in the name of gender specificity: the case of women are yet more examples, and not the main reason for Madame Du Châtelet's criticism.

Other problems remain in deciding the nature of an intentionally feminine, even feminist, polemic of the Examens de la Bible. Indeed, if the circulation of the work of Madame Du Châtelet is attested by numerous proofs, nothing allows us to say, in the current state of our knowledge, that Madame du Châtelet has intended to participate to the diffusion of this highly polemical work, or that she was aware, at the moment when she wrote this manuscript, of the existence of other manuscripts of the same nature. What is known about her life at Cirey, in the company of Voltaire, allows us to do no more than to elaborate a number of more or less plausible hypotheses, based on quite limited testimony, which is not always well informed 40 and which cannot for the present be compared with other sources: daily readings of the Bible, the composition of a very large work devoted to the scriptures, also polemical in nature.

However, neither our knowledge of Émilie's library, nor Voltaire's correspondence from this period provides us with any indication of the presence of clandestine literature within the Cirey coterie at the time at which the Examens were composed, while some of the most important works contained within this corpus (Examen de la religion, Traité des trois imposteurs, Analyse de la religion, Doutes sur la religion adressées au père Malebranche, and indeed the Testament de Meslier) were already being circulated and, in certain cases, widely disseminated. In fact, from a purely material perspective, Émilie would have had opportunity to read these texts even before her withdrawal to Cirey, in the salons that she frequented, for example, where she would have come into contact with some of the actors of the clandestine milieu, such as Fontenelle. But nothing in her biography or her public or personal writings allows us to assert this. There are, just five annotations of her hand on the manuscript of the Examen de la religion that belonged to Voltaire, and which is preserved today with the rest of his Library in St. Petersburg, but this unique case is very limited and does not allow us to draw general conclusions.

We may emphasize here a very small last point: a passage in which the author is revealed, perhaps, not in front of the reader, but in front of herself. When she comments on the story of Esther, the author of the Examens de la Bible gives us a brief reflection on the stupid education given to "Christian princesses", who are forced to learn the holy story by heart and without thinking, and who then feel compelled to imitate the bad example of the biblical princess who exterminated all those who did not think like her 41 was, in the eighteenth-century, the work of a woman, and which proposed to "involve women in all the rights of humanity, and especially those of mind." And Emily insists again: "This new education would be, in all things, a great benefit to the human race. Women would be better off and men would gain a new subject of emulation" 43 . May be, this interpretation is probably influenced by our current knowledge of the life and work of Madame Du Châtelet, but we must not forget that, despite the "feminist" scope of this preface, she remained in the manuscripts of Émilie until today and never circulated.

Nevertheless, the special status of the Examens de la Bible within the context of the clandestine corpus, and the provisional nature of our knowledge of them, will allow us to draw some key lessons in order to improve our understanding of this corpus and the place of the Examens in the intellectual history of the first half of the eighteenth-century. On the one hand, it would appear obvious that even though Émilie was unaware that she was participating in the creation of a highly influential intellectual movement, the reception given to her work reinforced her position as a "clandestine philosopherˮ. The manuscripts still extant (one copy in a private collection, one copy in the Royal Library of Belgium 44 and the collection held by the Troyes Public Library 45 , which also includes the Analyse de la religion chrétienne as well as the attached Notes and Preuves), reveal that the Examens de la Bible were not only disseminated in manuscript form during the eighteenth-century, but that they were specifically regarded as forming part of the clandestine family (in fact, B. Schwarzbach even suggests that the Examens may have influenced the author of the Preuves 46 ) even when the name of the author remained unknown or no more than a tradition that could not be corroborated. This intellectual proximity, the dissemination of the text, the aura and mystery associated with the years spent with Voltaire at Cirey, undoubtedly helped to forge the "legend of the manuscriptˮ with the Marquise engaged in secret combat against the church and its teaching. The image of the clandestine philosopher Émilie Du Châtelet is therefore, in reflection of the clandestine corpus, as much the result of a single, radical and highlyinformed work as a social and intellectual construct, created a posteriori by the possible intellectual proximity of the readers of the second-half of the eighteenth-century with the work -a methodological, critical and philosophical identity with other texts within the clandestine corpus. 43 It is the same of the "feminist" nature of its work. This character is not self-evident in the text, so much so that the receipt of the Examens will not have been very different from that of other anonymous works of this same corpus. The eighteenth-century readers However, even if we suppose that Madame du Châtelet had never read any of these clandestine writings, or that there was no reception, either direct or indirect, of her own observations on the Bible during the eighteenth-century (at the time when clandestine writings were being actively disseminated), the Examens de la Bible still bear witness to the existence of a state of mind shared by many of the other authors, including women, engaged in clandestine philosophical activity, in its methodological rigour, and in the strength of the rational criticism on which the text is based. This is precisely where one of the most unusual characteristics of the corpus of clandestine literature is to be found, which Émilie's manuscript also reinforces.

Conclusion:

This is undoubtedly the main lesson we can draw from the very special case of Madame Du Châtelet's Examens de la Bible : the clandestine philosophical literature is not only an intellectual phenomenon in itself, but the expression of a profound disruption across various sections of Ancien Régime society, from the aristocracy to the provincial petite bourgeoisie, from worldly society to places of residence far from the capital, from the highly masculine world of the learned and academic milieux, to the private world of a woman of reason, aspiring to become a "philosophe" like any other 48 .

There is one major lesson to be drawn from the work of Émilie Du Châtelet herself. She remains, to this day, the only female author of clandestine philosophical manuscripts that we have been able to identify. She is even, in all probability, the only woman to have written a 47 Ira O. Wade, Voltaire and Madame du Châtelet. An Essay on the intellectual activity at Cirey, Princeton, University Press, 1941 (réimp. New York, Octagon, 1967). 48 Paul Hazard, La crise de la conscience européenne (1680-1715), Paris, 1935. text of this nature, which is without a doubt one of the most methodical and most virulent directed against revealed religions and Catholicism in particular, most likely composed without any knowledge of the extent of the phenomenon of clandestine philosophy 49 . This fact provides confirmation, were it needed, of the extraordinary intellectual life of Émilie Du Châtelet which had an extensive impact on the scientific and philosophical output, both official and clandestine, of her time.

However, the second lesson is that the existence of this manuscript, within the context that I have attempted to describe briefly here, also shows us the scope of a phenomenon affecting every social class, every intellectual sphere, and which, indeed, also relates to a female audience. Although the case of Émilie Du Châtelet in her role as the author of this kind of text remains exceptional to this day, that of the reception of these writings among her female contemporaries is still a work in progress and has not yet been sufficiently explored, although it seems to me that it may still produce many surprises. No doubt therefore, Émilie du Châtelet occupies a major place as a "clandestine philosopher" and as a radical thinker of the French Enlightenment.

Maria Susana Seguin Université Paul-Valéry Montpellier III -IHRIM -UMR 5317 ENS de Lyon Institut Universitaire de France

  Condorcet and Turgot did not see in the text the work of a woman, but the legend of the collaboration of Emilie with Voltaire. It is also that the first critic could have conceived the text, following the research conducted by Ira O. Wade on the intellectual collaboration between Voltaire and Madame Du Châtelet during their stay at Cirey 47 . And even Bertram Schwarzbach will have needed a long and patient analysis of the text, in a critical edition way, to convince himself that the Examens de la Bible are the work of Émilie, and nothing but Émilie ...

  la littérature philosophique clandestineˮ, in Liberté de conscience et arts de penser. Mélanges en l'honneur d'Antony McKenna. Studies collected by Christelle Bahier-Porte, Pierre-François Moreau and Delphine Reguig. Paris, Honoré Champion, 2017, pp.77-90. 4 G. Lanson, "Questions diverses sur l'histoire de l'esprit philosophique en France avant 1750ˮ, Revue d'histoire littéraire de la France, 1912, 19, pp.1-29, 293-317. 5 I. O. Wade, The clandestine Organization and diffusion of philosophic ideas in France from 1700 to 1750, Princeton, 1938, republ. New York, 1967.

  . It is may be possible to recognize the same person who, translating Mandeville (another manuscript work of Madame du Châtelet) 42 , wondered why no great comedy, no great history, no great painting 40 On this subject, see the introduction by Bertram E. Schwarzbach already cited. 41 Examens de la Bible, p. 494. 42 Ana Rodrigues, « Emilie du Châtelet, a Bibliography » in Ruth Hagengruber (éd.), Emilie du Châtelet between Leibniz and Newton, Dordrecht, Springer, 2012, p. 207-246. See also Elena Muceni, « De la mauvaise éducation des filles », in La Lettre clandestine 27/2019, p. 215-237.

  Émilie du Châtelet, Préface du traducteur, Ms. Russian National Library of Saint Petersburg, Voltaire Collection, IX, pxx.

	My translation.
	44 Bruxelles-B.R. 15188-15189.
	45 Troyes-B.M. 2376-2377.

46

Bertram E. Schwarzbach, "Prefaceˮ, op.cit, pp.19-21.

The manuscript in question is Le philosophe, written by Dumarsais, and published for the first time in Nouvelles Libertés de penser, published covertly at Amsterdam in 1743. http://philosophie-clandestine.huma-num.fr/ms/144.

A copy of this manuscript is also preserved in the same volume as one of the copies of Madame Du Châtelet's Examens.

Ms Mazarine 1193, fol. 22-26v.

The publication of Nouvelles libertés de penser, a collection of clandestine texts, at Amsterdam, in 1743, opened a new era in the dissemination of clandestine philosophical literature which reached its fulfilment, during the 1760s, thanks to the editorial work of Voltaire and the circle around d'Holbach.


teaching and research institutions in France or abroad, or from public or private research centers.

The life of this author remains little known to this day. Born into a family of modest meanshis father was a paper merchant on the rue Saint-Denis in Paris -Boulanger demonstrated little enthusiasm for study during his youth. He nevertheless completed his studies at the College of Beauvais, under Jansenist influence, but did not study mathematics and architecture until later, teaching himself. He was also fascinated by ancient languages, and in particular oriental languages. In 1743, he was appointed private engineer to Baron de Thiers, who made him responsible, during the War of the Austrian Succession, for executing the excavation works during the siege of Freiburg im Breisgau. In 1745, Boulanger joined the Corps d'ingénieurs des Ponts et chaussées, under whose aegis he travelled throughout France, observing the terrain on which roads were to be constructed, often in harsh living conditions. He completed the construction of the Pont de Vaucouleurs, but his fragile health forced him to leave the construction site of the Pont de Foulain and to take early retirement from his post as engineer. He died soon afterwards, at the age of 37. During his visits to Paris, Boulanger associated with the milieu of the enlightened philosophers, with whom he collaborated. A close friend of Helvétius, who he called "Papaˮ and with whom he also corresponded briefly, he also contributed to the Encyclopédie of Diderot and d'Alembert, authoring the articles "Corvéeˮ, "Délugeˮ, "Guèbresˮ, "Hébraïque (Langue)ˮ and "OEconomie politiqueˮ. His most prominent works were quite widely distributed in manuscript form, and were finally published posthumously by Baron d'Holbach (in collaboration with Naigeon), in order to support the ideological struggle for the Enlightenment.

Primary sources:

[Diderot], "Extrait d'une lettre écrite à l'éditeur sur la vie & les ouvrages de Mr. Boulangerˮ, in L'Antiquité dévoilée par ses usages, 1766, pp. iii-xiv.

Works :

Recherches sur l'origine du despotisme oriental, s.l. [Geneva], 1761.

L'Antiquité dévoilée par ses usages, ou Examen critique des principales opinions, cérémonies et institutions religieuses et politiques des différens peuples de la terre, Amsterdam, Marc-Michel Rey, 1766

OEuvres mêlées, contenant différents articles extraits de l'Encyclopédie, savoir: Corvées, Déluges, Guêtres, Langue hébraïque, 1791 OEuvres de Boulanger, 10 vol., 1791 OEuvres de Boulanger, 6 vol., 1794. Recherches sur l'origine du despotisme oriental, édition Paul Sadrin, Paris, Les Belles-Lettres, 1988.

Dissertation sur Élie et Énoch:

This treatise was, in all likelihood, composed at the very end of Boulanger's life. His purpose is to "unmaskˮ an historical and theological deception and to reveal the true face of two imaginary figures on whom Scripture imposed the mythical persona of prophets: Elijah and Enoch. In the opening lines, he explains that Elijah is merely the embodiment of an astronomical symbol shown to the peoples after many years and centuries, and who was subsequently hidden, provoking the anguished expectation of the return of a fictional character to whom divinity was ultimately attributed. Boulanger supports his assertion using the customs of the Jews, the Bible and etymology. However, before coming to the case of Enoch, the Dissertation examines two figures that were not named in the title : the Roman Valerius Volusius and Ali, son-in-law and cousin of Mohammed, who are also presented as recurring symbols and emblems of the Great Judge. The goal of the treatise is unambiguous : the figures studied are but two examples selected from thousands of others, extending throughout geography and history, which confirm the universal character of human beliefs and the universality of the phenomenon by which primitive catastrophes are made the point of origin of the intellectual and religious history of mankind. The Dissertation ends with the study of Enoch, who merely confirms the same pattern ; the patriarch is simply an embodiment of the same principle, the illustration of the psychological mechanisms that lead to the development of religious beliefs.

Manuscripts:

Aix-en-Provence, B.M. 828 (844) Paris, Mazarine 1197 Saint-Petersburg, R.N.B. Voltaire 240 (IX).

Éditions :

Dissertation sur Élie et Énoch, ed. Paul Sadrin, Besançon / Paris, Les Belles Lettres, 1991. OEuvres mêlées, contenant différents articles extraits de l'Encyclopédie, savoir : Corvées,

Significance:

The works of Boulanger are those of a monomaniac. His works overlap, complementing and repeating each otherfrom the Nouvelle Mappemonde (1753) and his Mémoire sur une Nouvelle Mappemonde (1753) to his major treatises, Recherches sur l'origine du despotisme oriental (1761), L'Antiquité dévoilée par ses usages (1766), passing by way of the Dissertation sur Élie et Énoch and the articles published in the Encyclopédie. The reason for this all-consuming passion is grounded in science. Through his experiences as an engineer and field observations, Boulanger determined that the formation of the earth occurred over a considerable period of time. Moreover, the presence of various fossils in the layers of the earth attests to the historical reality of major geological catastrophes, preserved by tradition in the biblical story of the flood and the mythologies of almost every people on earth. According to Boulanger, our species, which almost perished in these primitive cataclysms, has lived continuously in a state of unconscious fear, obsessed by the idea that a new catastrophe might occur, destroying us forever. Every historical belief and activity of mankind, including its political forms, retains the mark of this primitive fear. Religion is not the product of divine revelation or political deception, but emanates from the primitive psychological trauma of humanity. It is this terror that explains the importance that human beings have continuously attributed to the natural cycles (day, lunar month, solar month, year, great year), which are enduring signs of death and resurrection. It is this terror that explains why, in all ages and places, religious institutions always offer a phase of anxiety followed by a phase of joy when the world, which appeared to be threatened with destruction, is "renewedˮ. This theory makes Boulanger an original thinker. Whereas the philosophers of the Enlightenment rely on a form of relativism in order to destroy the sacred character of the Christian religion, Boulanger, by contrast, demonstrates that Christianity as a religion resembles the others and that it is, for the same reasons as the other beliefs, a product of natural forces and of fear. The author also claims, following Bayle and Fontenelle, that the fables are not the product of a dysfunctional imagination, but that they speak to us about man, his concerns, his hopes, and his history.

Studies :

J. Hampton, Nicolas-Antoine Boulanger et la Science de son temps, Geneva, Lille, 1958. Jeroom Vercruysse, Bibliographie descriptive des écrits du baron d 'Holbach, Paris, 1971. Paul Sadrin, Nicolas-Antoine Boulanger (1722-1759)

  , Nicolas-Antoine Boulanger et la Science de son temps, Geneva, Lille, 1958. -Jeroom Vercruysse, Bibliographie descriptive des écrits du baron d'Holbach, Paris, 1971. -P. Sadrin, Nicolas-Antoine Boulanger (1722-1759) ou avant nous le déluge, Oxford, 1986.

  ou avant nous le déluge, Oxford, Voltaire Foundation, 1986. Maria Susana Seguin, Science et religion au XVIIIe siècle. Le mythe du déluge universel, Paris, H. Champion, 2001. -"Boulanger lecteur de manuscrits philosophiques clandestins?ˮ, (International Congress on the Enlightenment, Los Angeles, UCLA, 3-10 August 2003), La Lettre Clandestine 12, 2004, pp.121-134.-"Le traitement des sources érudites dans L'Antiquité dévoilée par ses usages de Nicolas-Antoine Boulangerˮ, La Lettre clandestine20, 2012, pp.169-185.

	Contributor:
	Maria Susana Seguin


Delphine Fradin

Rafael A Irizarry

Martin J Aryee

Peter Murakami

Thor Aspelund

Gudny Eiriksdottir

Tamara B Harris

Lenore Launer

Vilmundur Gudnason

Ph.D M Daniele Fallin
email: dfallin@jhsph.edu

M.D Andrew P Feinberg
email: afeinberg@jhu.edu

Personalized Epigenomic Signatures That Are Stable Over Time and Covary with Body Mass Index

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

Introduction

A role for epigenetics in common disease has long been suspected [START_REF] Bjornsson | An integrated epigenetic and genetic approach to common human disease[END_REF], and a strong relationship has previously been shown for cancer [START_REF] Feinberg | The history of cancer epigenetics[END_REF]. We have argued that common disease involves both genetic and epigenetic factors, and that epigenetic modification could both mark environmental effects as well as mediate genetic effects [START_REF] Bjornsson | An integrated epigenetic and genetic approach to common human disease[END_REF]. In addition to particular studies of exposure-epigenetic relationships, evidence of epigenetic changes with aging support an environmental component to epigenetic variation [START_REF] Boks | The relationship of DNA methylation with age, gender and genotype in twins and healthy controls[END_REF][START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF][START_REF] Christensen | Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context[END_REF][START_REF] Poulsen | The epigenetic basis of twin discordance in age-related diseases[END_REF]. Studies of identical twins show greater differences in global DNA methylation in older than in younger twins, supporting an agedependent progression of epigenetic change [START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF][START_REF] Poulsen | The epigenetic basis of twin discordance in age-related diseases[END_REF]. Previously we showed global methylation changes over an 11 year span in participants of an Icelandic cohort from the Age, Gene/Environment Susceptibility (AGES)-Reykjavik Study [START_REF] Bjornsson | Intra-individual change over time in DNA methylation with familial clustering[END_REF]. Recently, Christensen showed age-and tissue-related alterations in some CpG islands based on an array of 1,413 arbitrarily chosen CpG sites near gene promoters [START_REF] Christensen | Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context[END_REF], further supporting the evidence for dynamic methylation patterns over time. Other work, however, has suggested that epigenetic marks, or their maintenance, are themselves controlled by genes, and are thus heritable in the traditional sense and associated with particular DNA variants [START_REF] Heijmans | Heritable rather than age-related environmental and stochastic factors dominate variation in DNA methylation of the human IGF2/H19 locus[END_REF]. This would imply stability of methylation marks, rather than variation controlled by changing environments.

The work we will describe represents the first genome-scale, gene-specific analysis of DNA methylation in the same individuals over time. The results offer a solution to the conundrum of stable vs. time-sensitive differences in DNA methylation, as we will show both stable and dynamic sites of variable DNA methylation. Furthermore, the more stable sites of variation can identify a personalized epigenomic signature that may correlate with common genetic disease.

Methods

Samples. Non-immortalized lymphocyte samples were taken from participants of the AGES Reykjavik Study, which is described in detail elsewhere [START_REF] Harris | Age, Gene/Environment Susceptibility-Reykjavik Study: multidisciplinary applied phenomics[END_REF]. 74 samples contributed to these analyses. These reflect samples meeting our high quality array data filter among a randomly chosen set of 100 samples from the 638 AGES participants that had ample DNA from two visits. CHARM data were only considered in analyses if they passed our internal quality assessment.

For cross-sectional analyses of the most recent collection (visit 7), 64 samples contributed data, while 48 contributed to cross-sectional analyses of the earlier visit 6 data. For identification of dynamic VMRs, a subset of 38 samples had quality CHARM data at both time points. For the analyses with BMI presented here, BMI was calculated as the body weight in kilograms (kg) divided by the height in meters (m) squared.

Genome-wide methylation assay.

We performed comprehensive high-throughput array-based relative methylation (CHARM) analysis, which is a microarray-based method agnostic to preconceptions about methylation, including location relative to genes and CpG content [START_REF] Irizarry | Comprehensive highthroughput arrays for relative methylation (CHARM)[END_REF][START_REF] Irizarry | The human colon cancer methylome shows similar hypo-and hypermethylation at conserved tissue-specific CpG island shores[END_REF].

The resulting quantitative measurements of methylation, denoted with M, are log ratios of intensities from total (Cy3) and McrBC-fractionated DNA (Cy5): positive and negative M values are quantitatively associated with methylated and unmethylated sites, respectively. For each sample we analyze ~4.5 milion CpG sites across the genome using a custom designed NimbleGen HD2 microarray, including all of the classically defined CpG islands as well as all non-repetitive lower CpG density genomic regions of the genome. We include 4,500 control probes to standardize these M values so that unmethylated regions are associated, on average, with values of 0. CHARM is 100% specific at 90% sensitive for known methylation marks identified by other methods (e.g., in promoters), while including the more than half of the genome not identified by conventional region pre-selection. The CHARM results have also been extensively corroborated by quantitative bisulfite pyrosequencing analysis [START_REF] Irizarry | Comprehensive highthroughput arrays for relative methylation (CHARM)[END_REF].

Identification of VMRs.

We first screened the methylome for regions where methylation varied substantially across individuals. We term these variably methylated regions "VMRs", to distinguish them from regions identified for their discrimination of groups, such as tissue types or cases versus controls, which we and others have previously called DMRs. Our use of the term "VMR" can be considered a specific type of "metastable epi-allele" introduced by Rakyan in 2002 to denote variable expression of imprinted loci or variable methylation of an agouti methylation variant [START_REF] Rakyan | Metastable epialleles in mammals[END_REF].

To identify VMRs from our data, the raw CHARM data were first processed using the statistical procedure described by Aryee et al [START_REF] Ayree | Accurate genome-scale percentage DNA methylation estimates from microarray data[END_REF]. This statistical procedure produced quality metrics (percent between 0-100) for each sample and, for those that passed our quality test (>80%), a vector of methylation percentage estimates for each feature on the array. These were then smoothed to reduce measurement error using the standard CHARM approach [START_REF] Irizarry | Comprehensive highthroughput arrays for relative methylation (CHARM)[END_REF][START_REF] Irizarry | The human colon cancer methylome shows similar hypo-and hypermethylation at conserved tissue-specific CpG island shores[END_REF]. We denote the resulting methylation percentages for subject i at microarray feature j for time t as

Mijt.

We used cross-sectional analysis of visit 7 data to identify polymorphic variably methylated regions (VMRs) based on extreme inter-individual variance across consecutive probes. Specifically, we estimated between subject variability using the median absolute deviation (MAD), a robust estimate of the standard deviation [START_REF] Feinberg | Evolution in health and medicine Sackler colloquium: Stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease[END_REF]. We computed the median of |Mijt-mjt| across subjects, with mjt, the median Mijt across subjects i, and referred to it as sjt [START_REF] Feinberg | Evolution in health and medicine Sackler colloquium: Stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease[END_REF]. To avoid false positives in subsequent analysis of correlations with covariates, we required a very stringent definition for designating a polymorphic VMR: a region of 10 or more consecutive probes attaining values of sjt above the 99 th percentile of all the sjt and an average sjt > 0.125.

We chose these cut-off values using permutation tests. Specifically, we randomized the genomic order of the CHARM probes and applied the above algorithm to find VMRs (including the smoothing step) for each permuted data set. Using our criteria, we obtained 0 false positives. Lowering either the number of consecutive probes or the average sjt thresholds produced false positives. These VMRs were then annotated for genomic location and gene proximity. Genes within 3kb of VMRs were considered in a GO analysis of biological process categories. For each GO category, we performed a hypergeometric test [START_REF] Falcon | Using GOstats to test gene lists for GO term association[END_REF], with corresponding nominal p value, to determine enrichment of genes near VMRs. We also calculated the false discovery rate for each category statistic, to account for the multiple comparisons.

Identification of stable versus dynamic VMRs.

We generated methylation profiles for each sample using the average Mijt within the range of each VMR. This included a vector of k VMR values for each subject i and time point t. We calculated Dik, the median absolute within-person difference between methylation profiles from visit 6 to visit 7 for each VMR k. We then fit a two component Gaussian mixture model [START_REF] Banfield | Model-based Gaussian and non-Gaussian clustering[END_REF] to these values and used the resulting estimated posterior distributions to classify VMRs into three groups: "stable": those with posterior probability of membership in the lower distribution > 0.99, reflecting little intra-individual change over time; "dynamic": those with posterior probability of membership in the higher distribution >0.99, reflecting those with high intra-individual change over time; and "ambiguous": those not meeting either criteria, and thus in the overlap between the two distributions. (Note: Among the stable VMRs, there is some change over time observed in both directions, and when one takes the absolute value of this difference, the result is a small positive number, and thus the central tendency of Dk for stable VMRs is not zero.)

To evaluate discrimination of individuals based on patterns, we applied hierarchical clustering to the vectors of methylation values for the VMRs and graphed individuals into a dendogram based on similarity of VMRs. We then selected only those VMRs designated as "stable" in the analysis above and repeated the hierarchical clustering and dendogram graphic.

Identification of BMI-related methylated regions.

We performed cross-sectional analyses for data at each visit separately. For each stable VMR, we fit a linear regression model to summarize the relationship between BMI and methylation. Specifically, for each VMR k, we fit the following model:

Yi=ak + bkMik+eik
with Yi is BMI for individual i, Mik the methylation level for individual i in the k-th VMR, and e representing unexplained variability. Here bk represents the parameter of interest that summarizes the correlation between BMI and methylation. This produced one Wald-statistic for each VMR. We fit this model to the data from visit 7 and to account for the multiple comparisons due to multiple VMRs, we reported a list of regions with a false discovery rate of 0.30. To confirm these results, we independently applied the same regression approach to visit 6 and obtained estimates of b along with p-values.

Results

We report CHARM analyses on samples of the AGES study, assessing 4. time points, about 11 years apart, available for methylation analysis. We present data for 74 samples, a random set of those who had ample DNA remaining for both study visits. Descriptive statistics for these samples are given in Table 1.

We performed Comprehensive High-Throughput Array-based Relative Methylation (CHARM) analysis, which has been shown to identify differential DNA methylation without assumptions regarding where such changes would be, and uses arrays tiled through regions based on their relative CpG content, including all CpG islands, as well as CpG island "shores" which have been shown to be enriched in differential methylation {Feinberg, #47}. CHARM analysis in the visit 7 sample identified 227 regions meeting our criteria for polymorphic methylation patterns across individuals (variably methylated regions, VMRs). These represent regions of extreme variability across individuals defined by 10 or more consecutive probes with an average standard deviation > 0.125 (Supplementary Table 1). These VMRs showed enrichment for development and morphogenesis categories (Table 2), including genes from all four HOX clusters. The appearance of developmental genes is predicted by our model that epigenetic variation would involve developmental genes, and this variability itself increases evolutionary fitness in an environmentally changing world [START_REF] Feinberg | Evolution in health and medicine Sackler colloquium: Stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease[END_REF].

We then considered whether methylation at these regions changed within individuals over time.

The distribution of the absolute value of average within-person change in methylation over time per VMR is shown in Figure 1, which implies two underlying distributions. This fit a twocomponent mixture model, with 41 VMRs easily classified into the higher intra-individual difference group (probability of membership in orange distribution > 0.99, Figure 1), which we label "dynamic VMRs", 119 VMRs easily classified into the lower distribution (probability of green distribution > 0.99), which we label "stable VMRs", and 67 residing in the overlapping region and thus labeled "ambiguous" with respect to intra-individual change over time. Thus, approximately half the regions that are variably methylated across individuals appear to be stable over time within individuals.

These VMRs appear to act as an epigenetic signature. Figure 2A shows a dendrogram based on clustering of similarity in vectors of methylation values at VMRs. Use of only stable VMRs in the clustering algorithm uniquely identifies each individual (Figure 2B). These stable VMRs may represent polymorphic methylated regions that are not particularly susceptible to exposure modifications or that do not naturally change with age. One can postulate that these stable VMRs, sites that are variable across individuals but not due to exposures or age, may be the methylomic sites with variation most likely controlled by inherited mechanisms such as DNA variation or stochastic methylation reassignment in development.

Our results help focus epidemiologic hypotheses about how methylation may play a role in disease risk by allowing focused attention to particular VMRs in the methylome. In this vein, we explored the relationship between methylation and an easily measured phenotype, BMI, that is known to have many disease correlates. We identified 13 VMRs that met a false discovery rate (FDR) criteria of <25% in cross-sectional analyses of visit 7 (Table 3). Of these, 4 had a P < 0.10 and the same strength and direction of correlation with BMI at the earlier visit 6. These VMRs are in or near genes PM20D1, MMP9, PRKG1, and RFC5. To illustrate our findings, the methylation curves among obese (BMI≥30) and normal (BMI<25) subjects for the VMR at PM20D1 are shown in Figure 3. Scatter plots for the relationship between methylation and BMI for all four VMRs showing significant correlations at both visits are shown in Figure 4.

Discussion

We previously showed that global DNA methylation changes within individuals over time [START_REF] Bjornsson | Intra-individual change over time in DNA methylation with familial clustering[END_REF], and we have now confirmed and identified the locations of site-specific changes at "dynamic VMRs" using a genome-wide approach. In addition, we identified a separate set of "stable VMRs" that can be used to uniquely identify individuals, in a epigenetic signature akin to genetic fingerprinting. This signature may be correlated with disease status, implying epigenetic signature can mark disease risk or disease states. In particular, we show stable VMRs that correlate with BMI at two separate visits a decade apart. Some have argued that DNA methylation changes over time and is an important biological mediator of environmental effects on human disease, while others support the concept of inherited DNA methylation patterns, implying they are potentially variable across individuals but less likely to be dynamic over time. This has been a conundrum, since these appear to be opposing ideas. However, we show that both ideas have merit. It is important to identify these regions in the context of disease consequences, since those that are particularly labile may be the sites relevant when considering epigenetic marks as mediators of environmental effects, while those that are stable may be relevant as mediators or moderators of genetic effects.

Further, those that do not change over time can be used as an epigenetic signature for and individual, similar to genotype. These regions can then be considered as candidates for assessment of methylation associations with disease or health-related phenotypes under specific risk models.

Our results help focus the integration of methylation measurement into epidemiologic studies of disease risk by providing specific genomic sites for inquiry. Our exploration of possible correlations between methylation at these VMRs and an easily measured disease-related phenotype, BMI, identified 13 genes, 4 of which were consistently correlated with BMI across two separate study visits. Remarkably, many of these have been previously implicated in obesity or diabetes. MMP9, as well as another member of this family, MMP3, encodes a metallopeptidase that have been shown to be upregulated in obese individuals [START_REF] Nair | Increased expression of inflammationrelated genes in cultured preadipocytes/stromal vascular cells from obese compared with nonobese Pima Indians[END_REF]. Several MMPs, including MMP9, are upregulated in human adipocytes [START_REF] O'hara | Microarray analysis identifies matrix metalloproteinases (MMPs) as key genes whose expression is up-regulated in human adipocytes by macrophage-conditioned medium[END_REF]. Matrix metallopeptidases have also been previously associated with obesity in rodent models [START_REF] Uemura | Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress[END_REF][START_REF] Chavey | Matrix metalloproteinases are differentially expressed in adipose tissue during obesity and modulate adipocyte differentiation[END_REF]. Interestingly, PM20D1 is also a metalloproteinase, and, although not yet well-characterized, may have similar implications for obesity. PRKG1, a cGMP-dependent protein kinase, plays an important role in foraging behavior, food acquisition and energy balance [START_REF] Kaun | cGMP-dependent protein kinase: linking foraging to energy homeostasis[END_REF]. RFC5 is an intriguing gene as it encodes a metabolism-linked DNA replication complex loading protein, dysfunction of which leads to DNA repair defects. It might thus play a role in well-known but poorly understood DNA damage related complications of diabetes.

In an obese mouse model, SORCS1 has been located on a type 2 diabetes quantitative trait locus (QTL) [START_REF] Clee | Positional cloning of Sorcs1, a type 2 diabetes quantitative trait locus[END_REF], and this has been confirmed in humans, where SORCS1 SNPs and haplotypes were associated with fasting insulin secretion [START_REF] Goodarzi | SORCS1: a novel human type 2 diabetes susceptibility gene suggested by the mouse[END_REF]. IL1RAPL2 is located at a region on chromosome X that is associated with Prader-Willi like syndrome, while DACH2 is also an Xlinked gene associated with Wilson-Turner syndrome, both of which are Mendelian disorders with obesity features. TTC13 is part of a family containing another tetratricopeptide repeat gene, TTC8, that has been directly linked to Bardet-Biedl syndrome, which includes obesity as a primary feature. APCDD1 is a positional candidate gene associated with QTL that affects fat deposition in pigs [START_REF] Lee | Genomic sequence analysis of a potential QTL region for fat trait on pig chromosome 6[END_REF], and is located at a region on chromosome 18 that is linked to body fat (%) in men [START_REF] Lewis | Sex-specific findings from a genomewide linkage analysis of human fatness in non-Hispanic whites and African Americans: the HyperGEN study[END_REF].

Our identification of VMRs is of course limited by the number of individuals contributing to this genome-wide CHARM analysis. It is likely that increased sample sizes will improve detection of additional VMRs. Further, the dynamic VMRs defined here are based on an eleven year window among elderly participants. It is important to also identify methylomic regions that show intraindividual changes at early segments of the lifespan, and to connect these changes to particular environmental exposures. Also, our analyses were based on methylation patterns in DNA derived from blood, thus containing a mixture of cell types that could confound our results.

However, in our previous study of global DNA methylation (i.e. non-site-specific) in these samples, we found no relationship between lymphocyte count and methylation [START_REF] Bjornsson | Intra-individual change over time in DNA methylation with familial clustering[END_REF]. A recent paper by Heijmans (2010) also showed that cellular heterogeneity was not associated with DNA methylation amounts for the majority of sites they studied [START_REF] Talens | Variation, patterns, and temporal stability of DNA methylation: considerations for epigenetic epidemiology[END_REF]. Our use of blood as a DNA source may also limit the interpretations of these results, given the tissue specificity of DNA methylation. However, there is growing precedent for lymphoid tissues serving as a good surrogate tissue for changes in other target tissues. For example, loss of imprinting (LOI) of IGF2, one of the best studied disease-related epigenetic mutations, is found in both lymphocytes and colon, and changes of either are associated with increased colorectal cancer risk (Cui et al. Science 2003). Finally, our exploration of the correlation between BMI and methylation was based on availability of quantitative data and relevance to human disease. We were unable to assess the relationship of VMRs to categorical outcomes in this sample that is, although larger than previous genome-wide site-specific methylation reports, is limited for categorical phenotypes. This study supports further examination of other measures of obesity, diabetes, and related health consequences with respect to the particular VMRs identified here.

These results have important implications. An individual epigenetic signature that is stable over time has not previously been described. Such a signature could be driven by underlying sequence variation, by early environmental exposure, e.g. prenatally, or both. Even if in part or completely genetically driven, this epigenotype may be more proximate to the ultimate phenotype, in this case body mass index, and thus have considerable value for disease risk assessment. While the sample size is larger than previous genome-scale gene-specific methylation studies, it is still relatively small compared to classical sequence-driven approaches such as GWAS. Even so, the data suggest that this epigenomic approach to disease phenotype will be an important complement to such studies. Even with these numbers, we could identify four genes with VMRs related to BMI. In addition, the identification of stable VMRs may have long term consequences for developing personalized epigenomics in medicine, that might reflect both one's genome and early (e.g. in utero) environment.

  5 million CpG sites genome-wide. In brief, the AGES study constitutes visit 7 (in 2002-2005) of the Reykjavik Study, which began with 18,000 residents of Reykjavik recruited in 1967. The AGES study recruited 5758 of the surviving members, who were aged 69-96 years in 2002. Of these, 638 gave a DNA sample in 1991 as part of the sixth Reykjavik Study visit, and therefore have DNA from two

Figure 1 :

 1 Figure 1: Distribution of intra-individual change over time at VMRs. Mixture distribution analysis shows Dk, the average absolute value of intra-individual differences in methylation over time, fits two underlying curves: little change, "stable" in green and larger changes, "dynamic" in orange.

Figure 2 :

 2 Figure 2: Similarity between individuals based on VMR methylation. Panel A: Dendogram based on clustering applied to methylation profiles at all 227 VMRs. Panel B: Dendogram based on clustering applied to methylation profiles using only the 119 stable VMRs. Numbers represent individual IDs.

Figure 3 :

 3 Figure 3: Methylation Curves at PM20D1. Methylation curves for visit 7 and visit 6 data. Dashed lines are individual methylation curves. Solid lines are average curves by obese (blue) and normal (red) groups. The green line indicates the boundaries of the VMR. CpG density is shown below with CpG islands marked in orange. Gene location shown at bottom.

Figure 4 :

 4 Figure 4: Correlations Between Methylation and BMI at Six BMI-Related VMRs. Points are individual IDs, blue indicates visit 7, red indicates visit 6.

Table 1 . Descriptive Information (Mean (standard error)) for Samples Used in CHARM Analyses at Each Time Point

 1

	Visit 6	Visit 7
	(1991)	(2002-2005)

Table 2 . Gene Ontology Results with P<0.01 for 227 VMRs Identified

 2

		Odds	Obs	Expected
	Pvalue FDR	Ratio	Count	Count	GO Term	Genes
	0.0011 0.222	7.04	5	0.79	Ant./post. pattern formation	HOXA5; HOXB6; HOXD8; HOXC10; HOXA1
	0.0019 0.222 43.31	2	0.07	blastoderm segmentation	HOXB6; HOXD8
	0.0019 0.222 43.31	2	0.07	determ. anterior/post. axis, embryo	HOXB6; HOXD8
	0.0082 0.256 17.31	2	0.14	neuron recognition	FOXG1; NTM
	0.0086 0.256	3.63	6	1.77	pattern specification process	HOXA5; FOXG1; LEF1; HOXC10; MYF6; HOXA1
	0.0096 0.256	7.47	3	0.44	placenta development	ESX1; LEF1; CDX4
	0.0096 0.256 15.74	2	0.15	intra-Golgi vesicle-mediated transport COPZ1; GABARAPL2

Table 3 . Stable VMRs Associated with BMI

 3 Bold values indicate confirmation in visit 6 analysis (p<0.1 and consistent regression parameter estimates); italics indicate conflicting directions of correlation with BMI

				Visit 7		Visit 6
		Nearest			Regression		Regression
	Chr	Gene	Qval	Pval	Estimate	Pval	Estimate
	chrX	IL1RAPL2	0.114	0.00304	-20.3	0.266	-8.9
	chr1	PM2OD1	0.114	0.00332	7.6	0.00824	7.7
	chr6	NEDD9	0.114	0.00351	12.1	0.38	5.2
	chr20	MMP9	0.160	0.00658	11.6	0.0605	8.9
	chr10	SORCS1	0.215	0.0128	-13.6	0.112	-9.4
	chr10	PRKG1	0.215	0.0132	11.8	0.000711	18.9
	chr12	RFC5	0.243	0.0175	-11.8	0.0653	-8.8
	chr1	TTC13	0.249	0.022	9.27	0.523	3.3
	chrX	DACH2	0.249	0.0311	-15.1	0.539	4.1
	chr5	TRIM36	0.249	0.0326	11.3	0.0781	-14.1
	chr14	FLRT2	0.249	0.0278	-9.5	0.19	-5.8
	chr1	C1orf57	0.249	0.0253	-10.6	0.282	-6.5
	chr18	APCDD1	0.249	0.0332	-10.7	0.901	0.7

Acknowledgments

This work was funded by the NIH grants 1R01ES015211 awarded for Dr. Fallin and 2P50HG003323 awarded to Dr. Feinberg. The AGES Reykjavik study is funded by NIH contract, N01-AG-12100, Hjartavernd (the Icelandic Heart Association), and the Althingi (the Icelandic Parliament).

Author Contributions

M.D.F., V.G., A.P.F. designed the study; D.F. performed CHARM assays; D.F., R.I., M.J.A., P.M., T.A., performed statistical analyses; V.G., G.E., T.H., L.L. designed the AGES study and recruited participants; D.F., R.I., M.D.F., A.P.F. interpreted the data and wrote the paper.