Ines Di Loreto
email: ines.diloreto@fastwebnet.it

Fabien Hervouet
email: fabien.hervouet@lirmm.fr

Identity: the Contribution of Contemporary Philosophy to Agent Design

This paper aims to present some perspectives about the agent design research community. Typically in perspective paper we try to understand what the trends are in order to foresee possible future directions. Exceptionnally, instead of looking at where the agent community is going analyzing numerical trends, with this article we want to suggest where agent research could go but is not yet going. During our analysis we discovered that a large part of the agent research community based its work on particular concepts derived from western analytic philosophy based on the logical approach. While applying a pure logic approach could be enough to model interactions for purely artificial agents, we believe that with the advent of mixed environments -i.e. environments involving both humans and agents -we have to focus on a more human-compatible agent design. For this reason in this paper we analyze the identity concept in both, agent design and philosophy focusing on uniqueness, mind, and body, in order to show that other paths could be studied in order to create different directions in agent design.

Introduction

This paper aims to present some perspectives about the agent design research community. Typically in perspective paper we try to understand what the trends are in order to foresee possible future directions. Exceptionnally, instead of looking at where the agent community is going analyzing numerical trends, with this article we want to suggest where agent research could go but is not yet going.

We are of the opinion that in the agent research community most of the current trends originate from the translation of particular concepts -mostly from analytic philosophy -which are only a particular western way to look at philosophy and agents. With this article we want to suggest that other paths originating from philosophy could be taken into account in order to create different directions in agent design. It is obviously not the aim of this paper to exhaustively explore over 3000 years of philosophical research nor 60 years of agent design. For this reason the paper will focus only on 20th century philosophy and will only deal with the concept of identity in both agent design and philosophy. In fact while analyzing current trends we observed that while trying to model and reproduce humans and societies, agent design mostly does not use a structured construction of the identity concept.

This paper will not only show why a part of contemporary philosophy has already abandoned the dualism of mind/body but also that contemporary philosophy has an interesting way of considering the concept of uniqueness, performance and environment as strictly linked, interacting with each other. This philosophical analysis will clearly be of no interest to the agent research community if not linked to a parallel analysis of trends in agent design research. For this reason the core part of this paper is structured to compare the state of the art of the concept of uniqueness, body, and mind in the agent research community and in parallel in philosophy.

After the analysis conducted for this paper, we feel that a major part of the agent research community has fallen into the "utilitarian trap" by considering agents as artificially disconnected from our human world. We are of the opinion that applying a pure logic approach could be enough to model human-like interactions in a purely artificial agents environment. On the other hand, because of the advent of mixed environments -i.e. environments involving both humans and agents -we think we have to focus on a human-compatible agent design. This is mainly what motivated the double study -agent design / philosophy -we conduct in the rest of the paper.

The role of identity: from philosophy to agent design

In order to understand what importance the concept of identity could have in agent design we will start by analyzing the role of the concept of identity more generally in computer science. In the last few years -with the widespread popularity of social networks -the classical idea that suggests a perfect -but very simplified -match between identity and user name has been questioned. Today it is widely accepted that identities are negotiated based on the individuals' characteristics as well as on the community's state and aims [START_REF] Wenger | Communities of Practice: Learning, Meaning, and Identity[END_REF]. From this point of view, the participants' positioning is based not only on individual moves, but on phenomena that are both context-shaped (e.g., the community/social network I decided to join) and context-renewing (built throughout each interaction) [START_REF] Schegloff | Reflections on quantification in the study of conversation[END_REF]. The awareness of the importance of identity so well identified in psychology and social network analysis seems to have little impact on agent design, even if their stated purpose is to recreate some kind of human behavior. A possible criticism of the translation of this approach to agent design is that while any human individual's existence cannot be replaced by any other, and therefore does not fall into any general identity, in the multi-agent world an agent is a general concept -i.e. a generic piece of code -and can a priori be replaced by any other agent. While this abstraction is not false, this kind of uniformity can create problems when trying to model agents with human behavior interacting with human beings. Is it really true that the same starting point should have the same ending point passing through the same interactions?

In order to understand the importance of this question we want to underline the already known fact that concepts are not neutral. For example, based on the way the two concepts of man and woman are conceived and linked to the sex equality concept they can lead towards two very particular societal systems.

On one hand Mary Wollstonecraft [START_REF] Wollstonecraft | A Vindication of the Rights of Woman (Norton Critical Editions)[END_REF] -one of the founding feminist philosophers -theorized a society "controlled and organized in asexual forms" which does not take into account the membership of the citizen of one sex or the other. In this kind of society, there are no men and women, only "Universal Men"1 . The cult of the universal is expressed through the reduction of sensible bodies and individual differences to the "One and Universal Man", also implying the equality of the sexes in the particular way seen above. In this kind of abstraction there are two negations: the negation of the Body (and thus differences in sex), and the negation of the Individual.

Wollstonecraft's point of view could be compared with that of Olympe de Gouges [START_REF] De Gouges | La musa barbara. Scritti politici (1788-1793[END_REF] who foresaw a society where men and women are not reduced to a single subject neutral and disembodied (precisely because disembodied is neutral). Women, she says in her "Declaration of Rights", take part in the founding of the state precisely because they are a "different subject".

It is evident that these two points of view imply a different use of the same general concepts which lead towards different societies.

Bringing the analysis back to agent design we believe that the oversimplification of the concept of identity is caused by the fact that computer scientists -and thus agent designers -based their work on the "Universal Agent" concept promoted by philosophy. The rest of this paper will analyze if and how the identity concept can be developed differently, paralleling agent design and contemporary philosophical assumptions about the con-cepts of uniqueness, body and mind, which are fundamentally negated by the use of the "Universal Man" concept.

The concept of uniqueness

As far as we know, uniqueness is a concept that has been studied in philosophy without entering the computer science sphere. This section presents possible reasons of this impermeability in the agent design community and explains recent philosophical theories computer scientists could try to integrate in their research.

The role of uniqueness in agent design

There is almost no debate about uniqueness in agent design. More generally uniqueness is in essence an issue for computer science. The transition from the analog era to the digital age has brought about the notion of copy.

Copying is an intrinsic property of digital data. Any data can be copied and replicated with an absolute guarantee of ending up with two exact similar objects. This interesting property prevented researchers from really tackling such an issue.

Practically, in computer science, labels are the way we usually handle identity. In a computer language what we call variable is just a name referencing a specific memory space containing a piece of information. Data is stored in the memory and we can access it using suitable human-understandable names. Similarly, in multi-agent systems agents are uniquely handleable by their generated names, from the omniscient point of view.

Moreover, in MAS identity is mainly structured from the point of view of the role of the agents. Thus, MAS usually put a multitude of agentsidentical for each role from the source code point of view -together in order to accomplish a certain global task or to have a certain global behavior. This means that even if agents do not act exactly the same in a local way, they often originate from the same piece of code that takes into account their interaction with their peers.

However, there is one research topic which addresses the severe lack of uniqueness in MAS: the trust problem. Indeed, in multi-agent systems each agent needs to be pretty sure of who it is dealing with especially if, for instance, it is dealing with economic decisions. In such a "hostile environment" the agent's goal is to choose its future interaction partners and shape these interactions based on its personal experience with the peers. The implemented techniques used in this case are the same used by human agents in online social communities, such as reputation (in the sense that all other agents may evaluate or devalue an agent based on its past interactions with him). See [START_REF] Ramchurn | Trust in multi-agent systems[END_REF] for a general introduction on this issue and its associated challenges.

Another interesting point of view of the concept of identity/uniqueness applicable to computer science, comes from two biologists, Maturana and Varela, who addressed the complex problem of autonomy, knowledge and identity in [START_REF] Maturana | Autopoiesis and Cognition: The Realization of the Living[END_REF]. In this work they characterized living organisms by coining the concept of autopoiesis. They defined autopoiesis as a complex process of self-production of the system by itself. Thus, an autopoieitic system should be seen as a machine that continuously generates and specifies its own organization. It accomplishes this incessant process of replacing its components because it is continuously subjected to external disturbances and constantly forced to compensate for these disturbances. In short an autopoietic system can be seen as a homeostatic system whose invariant principle is its own organization (seen as the network of relationships that defines it). Even if the authors don't use the term of uniqueness in their book, they clearly claim that identity is the product of the historical coupling between the organism and its environment. Therefore, uniqueness may be defined as this historical coupling, i.e. the historical adaptive activity of the structure in order to fit the organization. The autopoiesis theory has inspired some scholars in the artificial life and agent design domains such as [START_REF] Bourgine | Autopoiesis and cognition[END_REF][START_REF] Beer | Autopoiesis and cognition in the game of life[END_REF][START_REF] Ziemke | Are robots embodied?[END_REF], but it is generally very few addressed.

The role of uniqueness in philosophy

As we have seen from this short overview of the uniqueness concept, while seeing uniqueness as a problem most part of agent design tries to resolve it using a "labeled" approach (i.e. designing uniqueness only as a minor agent's attribute). As a result a certain part of agent design seems to work on a "Universal Agent" concept which does not need a structured uniqueness to be implemented. In addition the most interesting and structured point of view on uniqueness comes from "outside" the computer science domain. Similarly in philosophy there are different points of view regarding the concept of "Universal" man, and different criticisms which could help to open new paths in agent design. In particular feminist philosophers of the second wave did the more interesting work in destroying the predominance of the concept of "Universal" identity. Hannah Arendt before and Adriana Cavarero after, accused philosophy of constructing a universal "science" on the definition of "Man" declaring the uniqueness of each human being not "scientifically" defined and therefore unnecessary to his real existence in the world. Philosophy in fact always asks what is a supposed universal reality (the Man, the Being, the Subject, etc..) and ignores the other important question that living beings deal with one another: who are you?.

In [START_REF] Adriana Cavarero | Le filosofie femministe[END_REF], Adriana Cavarero analyzes the important role played by Christine Battersby in defining the concept of identity. The philosopher focuses on a concept of personal identity consisting in a game of relationships that makes the identity smooth, resonant and dynamic. For Battersby [START_REF] Battersby | The Phenomenal Woman: Feminist Metaphysics and the Patterns of Identity[END_REF] identity it is not fixed and permanent as in the metaphysical tradition which loves to look at it as a "substance". It is not even the mere whirl of countless fragments as most of the contemporary feminist philosophy wishes it to be. Instead it is the identity of a relational self that persists without being the same, and becomes a fluid motion and permeable in its persistence. Apart from social, biological, and philosophical consideration the challenge of designing a self this way is radical because the self is thought of as relational -with other human beings but also with things -as well as a singularity, a uniqueness.

While uniqueness is a "new concept" in the long history of philosophy, psychology has discussed it from the very beginning of its history. In the '60s, Piaget led the constructivist movement, mainly in response to behavioral theories dominant at this time, considered as too simplistic. His vision is that every individual has the ability to hold their own reconstruction of reality. This educational philosophy has been radicalized by von Glasersfeld, who refutes the notion of external or ontological representation of reality. He introduces the viability concept, which is a relationship closer to the experience situated between knowledge and reality [START_REF] Glasersfeld | Pourquoi le constructivisme doit-il être radical ?[END_REF]. What he considers viable could be any action, a conceptual structure, a theory as long as it is useful to complete a certain task, to achieve a goal. He agrees with Piaget when saying that knowledge doesn't only aim to copy reality but it is more used for self adaptation.

This psychological movement, as a theory of knowledge, supports the fact that identity is perpetually in construction, deriving from our own adaptation process in direct confrontation with the environment. This point of view will be explored further in the '80s by Varela, Rosch and Thompson and their theory of enaction. In [START_REF] Varela | The Embodied Mind: Cognitive science and human experience[END_REF], the authors reintroduced the notion of one subject's unique experience as the center of the problem. The enaction paradigm postulates the co-emergence of both cognition and perceived world through the performative body in action in the environment. The authors also refuse any pre-given representation in favor of a complete construction of an embodied cognition. Each identity is then performative and unique, based on the subject's interactions. Therefore identity -and uniqueness with it -becomes a pure bottom-up mechanism that conflicts with the top-down approach.

The concept of body

The second aspect implied by the "Universal Man" concept is the abstraction from the body. As a matter of fact computers are not embodied, nor are computer programs. In this section we will see why and how the concept of body has been discarded by agent design researchers, because of the disembodied philosophical conception.

The role of the body in agent design

From the very beginning of computational science, the body hasn't been a priority at all. In essence calculus is disembodied as it is a pure mathematical abstraction, and computer science borrowed the same approach. However, regarding the "body concept" it is very interesting to notice a fundamental difference in the agent design approach between two domains: computer science and robotics. Indeed, while computer science focused mainly on disembodied reasoning capabilities, robotics was created with the idea of body, of physical interaction, and it is based on the experimental principles of physics and mechanics in a very grounded manner. Therefore, the advent of robotics is a major step towards the consideration of the notion of embodiment for intelligent agents, even if the transition from the automaton view to the agent view did not happen immediately in robotics, heavily influenced by the control engineering.

As Tom Ziemke argues in [START_REF] Ziemke | Are robots embodied?[END_REF], it has been recognized for a decade that embodiment is a necessary condition to characterize living organisms. More and more researchers have attempted to address this absolute need for embodied cognition (see for example [START_REF] Pfeifer | Understanding intelligence[END_REF][START_REF] Varela | The Embodied Mind: Cognitive science and human experience[END_REF][START_REF] Clark | Being There: Putting Brain, Body, and World Together Again[END_REF]). Ziemke also says that despite a general acknowledgement, robotics "is largely 'stuck' in the old distinction between hardware and software". He clearly claims that Searle's famous Chinese Room Argument (CRA) [START_REF] Searle | Minds, brains, and programs[END_REF] -which says in short that disembodied manipulation of symbols cannot be considered as an authentic and intelligent understanding -remains unsolved. The main reason for this claim [START_REF] Ziemke | Rethinking grounding[END_REF], is that the cognitivism vision -seeing the mind as a computational tool, i.e. manipulating symbols and rules, completely disconnected from any physical reality -is still dominant over the enaction vision.

However a kind of reactualization of the CRA has been proposed by Stevan Harnad [START_REF] Harnad | The symbol grounding problem[END_REF] and its Symbol Grounding Problem (SGP). The SGP questioned the research community in a more precise way: will an artificial agent one day be able to develop a semantic autonomous capability allowing it to establish its own semiotic networks connecting some of its own symbols to the environment it evolves in? A lot of researchers have worked on this problem, especially Luc Steels who was a pioneer with his Talking Heads experiment [START_REF] Steels | The Talking Heads Experiment[END_REF].

Nevertheless the real revolution in robotics has occurred mainly within the last decade, during which we have seen the rise of the new discipline of developmental robotics. This field lies at the intersection of a number of scientific and engineering disciplines including at least developmental psychology, cognitive science, artificial intelligence, robotics, and philosophy. The key idea of developmental robotics is "physical embodiment" which considers the body as something essential in the realization of cognition and action. Developmental robotics explains that the body specifies the con-straints on the interaction between the agent and its environment. Thus, research is axed on the sensorimotor mapping at the early development stage, and on the following stage of social development, aiming to fill the gap between these two phases in human life. See [START_REF] Asada | Cognitive developmental robotics: a survey[END_REF] for a recent survey of this quite young but very promising research domain.

The role of the body in philosophy

As we did for the uniqueness concept in the previous section, in this section we explore the idea of body from a philosophical point of view to see if we can add some interesting concepts to our computer scientist point of view.

The first concept we will analyze is the one of "constructed body". Judith Butler [START_REF] Butler | Bodies That Matter: On the Discursive Limits of Sex[END_REF][START_REF] Butler | Gender Trouble: Feminism and the Subversion of Identity[END_REF] states -on the wave of Simone de Beauvoir but also Monique Wittig -that not only gender is socially constructed but also the body is socially constructed. What the author means with this statement is that not only can a body be physically manipulated but also that gender, sex and body prove to be performative, capable of building identity through performance. For the above mentioned authors, identity is constructed through both a performative mental approach and a performative bodily approach. At the core of this statement there is the request to abandon the Cartesian (but also Platonic, Kantian and the like) mind / body dualism. In addition, the body is the instrument of our contact with the world, namely the world takes on a different aspect according to our way to grasp it. The body-object described by scientists exists only in the concrete experience from the subject, and is not an abstract entity. However, because of this idea the "body" often appears to be a passive medium that is signified by an inscription from a cultural source figured as "external" to that body. It is exactly this kind of separation that agent design borrowed from philosophy.

But what does having a performative body mean? In Judith Butler's article, "Performative Acts and Gender Constitution: An Essay in Phenomenology and Feminist Theory," the author argues that philosophers rarely think about acting (in the theatrical sense of the term) -this is more the habit of psychologists -but they do have a discourse of "acts" that maintains associative semantic meanings with theories of performance and acting. For example, John Searle's 'speech acts' seem not only to refer to a speaking relationship but to constitute a moral bond between speakers. Further, 'action theory', a domain of moral philosophy, seeks to understand what it is 'to do' prior to any claim of what one ought to do. Finally, the phenomenological theory of 'acts', by Edmund Husserl, Maurice Merleau-Ponty and George Herbert Mead among others, seeks to explain the mundane way in which social agents constitute social reality through language, gesture, and all manner of symbolic social sign [START_REF] Butler | Performative acts and gender constitution: An essay in phenomenology and feminist theory[END_REF]. However, even in this case, they seem to assume the existence of an agent existing prior to performances and language.

When Simone de Beauvoir claims, "one is not born, but, rather, becomes a woman," [START_REF] De Beauvoir | The second sex.Translated from the French and[END_REF] she is appropriating and reinterpreting this doctrine of constituting acts from the phenomenological tradition: identity is created through the subject performances/acts. However for her there is no stable a priori identity from which various acts proceed; rather, it is an identity tenuously constituted in time -an identity instituted through a stylized repetition of acts. A dynamic identity where the body is both, subject and object of the construction. Not only is identity both body and mind, but like for the uniqueness concept it is dynamically constructed in an environment. Once again we are facing an approach pushing towards a bottom-up identity contruction. The presence of a human being in the world strictly implies the presence of a body that is both a thing of the world and a point of view over the world. Donna Haraway [START_REF] Haraway | A Cyborg Manifesto: Science, Technology, and Socialist-Feminism in the Late Twentieth Century[END_REF] will push this interpretation to claim for a condensed image of both imagination and material reality -the two joined centers structuring any possibility of historical transformationachieved through the cyborg's image. In addition the body is relational. The question who are you? that we mentioned before then takes on a special connotation. Every human being is unique because by simply exposing himself to the gaze of others, he is already unique in the body shape and tone of voice [START_REF] Adriana Cavarero | Le filosofie femministe[END_REF], even before his name is known. That is even before the label we attribute to him. Existence is therefore this mutual exposure in a shared space where everyone, from birth is unique, and in the course of his life can show who he is with acts and words.

In the previous section, uniqueness was defined as a historical coupling, i.e. the historical adaptive activity of the structure in order to fit the organization. In this section we are proposing a similar definition based on the interaction body/environment. While computer science looks at the body as quite a stable "hardware" element, philosophers suggest that the body is both a complex cultural construct, and a physical object interacting with the world. The body then is not a tool through which the mind manipulates the world, and it is not an attribute of the mind. On the contrary it is a historical adaptive coupling.

The concept of mind

As previously said, if the "Universal Man" is a disembodied entity then the mind and its thoughts become the central part of philosophical and scientific reasoning. This section is devoted to analyze this kind of approach and its criticism.

The role of the mind in agent design

From the historical point of view, we can easily claim that agent design started with the birth of cognitive sciences with the Macy's conferences in 1946. We can also bring back at this time the birth of the conceptual gap of how to consider human cognition -represented by the two famous researchers John von Neumann and Norbert Wiener -even before the construction of the first computers. On one hand, von Neumann considered cognition as fundamentally oriented toward problem solving, while Wiener claimed that cognition is more like an autonomous and self-creative activity, which he thought more credible to describe living systems. von Neumann's vision has been widely promoted and become dominant, giving birth to computer and engineering sciences. Besides this, computer is also the dominant metaphor used to design the brain, which is mainly based on the idea of an information process from inputs to outputs [START_REF] Ziemke | The construction of 'reality' in the robot: Constructivist perspectives on situated artificial intelligence and adaptive robotics[END_REF]. The other idea of autonomous aspects, coming from living systems, has been almost completely neglected. According to [START_REF] Maturana | Autopoiesis and Cognition: The Realization of the Living[END_REF], von Neumann advocates the heteronomous systems, while Wiener and his cybernetics advocates the autonomous systems. In other words, heteronomous systems think in terms of I / O, while autonomous systems think in terms of operational closure. Always according to [START_REF] Maturana | Autopoiesis and Cognition: The Realization of the Living[END_REF] while heteronomous systems are in a representational relationship with the environment, autonomous systems are in a meaning-emergent relationship with the environment. This implies two different logics: the logic of correspondence for heteronomous systems and a logic of consistency for autonomous systems.

For a very long time agent design has only taken into account the omniscient point of view. Indeed at the beginning, researchers thought it was a great opportunity for the computer to know exactly the whole system data. Thus they applied this omniscient strength to their algorithms. As an example, just think about how the A ⋆ algorithm tries to solve the problem of moving from one point to another for a situated agent through graph theory, from a pure omniscient point of view. This kind of approach is quite similar to the philosophical one we talked about in the previous section, and which perceives the agent as en external abstract entity. While the algorithm is effective, it is a good example of the predominance of the mind over the body, even when talking about issues like displacement involving the body directly.

Furthermore, for a large majority of multi-agent frameworks, the environment is only seen as an opportunity of communication between agents. Besides, in agent design it is very interesting to inspect the way we make agents communicate. Communication is a root mechanism in computer science -just think about the case of message passing in the object oriented paradigm. Thus there was then no reason not to also provide soft-ware agents with a communication mechanism. Thus in the '90s two main standards emerged in multi-agent systems in order to formalize communication: KQML and FIPA-ACL. These standards are philosophically based on Austin's and Searle's theories about speech acts, profoundly rooted in a pure mind-only logic. This is another example of the predominance of the mindfully dominant vision in the artificial intelligence community.

Nowadays, although the metaphor of the agent as a symbol interpreter is always present, things became more complex. For instance, Russell and Norvig's proposed a more complex approach to this issue. In their modern approach to artificial intelligence [START_REF] Russel | Artificial Intelligence: a modern approach[END_REF] -which is considered as a major reference in the AI teaching and research community -they distinguish four kinds of agents: reactive agent (which selects an action from its current situation and a set of rules), model-based agent (which extends the reactive agent by having a memory), goal-based agent (which tries to reach a state usually using planning), and utility-agent (which tries to maximize its satisfaction by trying to reach the most satisfying state). In the same movement we can also cite the Belief-Desire-Intention model [START_REF] Bratman | Intention, Plans, and Practical Reason[END_REF] which is a more complex model articulated around the notion of knowledge: beliefs represent the information (true or false) the agent has about the world, desires represent long term goals the agent would like to accomplish, and intentions represent short term goals according to the desires.

In 1986, Minsky -after long years of collaboration with Papert -proposed a different perspective in his Society of Mind [START_REF] Minsky | The society of Mind[END_REF]. In this book he presented a cognitive model in which the mind is considered as a distrubuted multi-agent system with local and global adaptation. These agents are organized into agencies which can also be organized in network of agencies. His approach was very innovative and considered by Varela as a middle path of cognitive science between centralized cognitivism and distributed autoorganization [START_REF] Varela | The Embodied Mind: Cognitive science and human experience[END_REF]. However, even in these cases we faced with mind-centered approaches.

As we discussed in the previous section, the importance of the body was put back into the ring thanks to robotics. But robots are just embodied agent who also need to be designed with a mind. This is the reason why the way robots interact with their environment has been thoroughly studied for fifty years. Three major kinds of robotic architectures emerged. Deliberative architectures were historically the first ones to be proposed [START_REF] Nilsson | Principles of Artificial Intelligence[END_REF][START_REF] Giralt | An integrated navigation and motion control system for autonomous multisensory mobile robots[END_REF]. These architectures use symbolism and are generally organized into multiple hierarchical layers, each layer communicating only with its direct superior and/or inferior neighbor layer. But these deliberative architectures lack responsiveness. In direct opposition, purely reactive architectures were considered [START_REF] Brooks | A robust layered control system for a mobile robot[END_REF][START_REF] Agre | Pengi: An implementation of a theory of activity[END_REF][START_REF] Arkin | Motor schema based navigation for a mobile robot: An approach to programming by behavior[END_REF]. They are built by stacking finite state machines called behaviors, directly connecting sensors to actuators in a completely reactive way. They focus on the idea that complex behavior can emerge from a composition of simple reactive behaviors, without reasoning nor symbolization. Moreover, a few years later Brooks released two major articles [START_REF] Brooks | Intelligence without representation[END_REF][START_REF] Brooks | Intelligence without reason[END_REF]. The first one was an accusation against the notion of representation on which AI is based. In the second one he put forward the many benefits of bio-inspired architectures. Subsequently hybrid layered architectures [START_REF] Firby | Adaptive Execution in Complex Dynamic Worlds[END_REF][START_REF] Gat | Integrating planning and reacting in a heterogeneous asynchronous architecture for controlling real-world mobile robots[END_REF][START_REF] Bonasso | Experiences with an architecture for intelligent, reactive agents[END_REF][START_REF] Alami | An architecture for autonomy[END_REF][START_REF] Luzeaux | Harpic, an hybrid architecture based on representations, perception, and intelligent control: A way to provide autonomy to robots[END_REF] have been developed in order to combine the advantages of behavioral and deliberative architectures. Thus they have both a hierarchy of layers dedicated to symbolization and decision making as well as reactive nested loops allowing each layer to provide appropriate responses to the dynamics and the operational reality of the robot.

The role of the mind in philosophy

In the 20th century problems deriving from the division between mind and body also become evident in philosophy. One of the most well-knownand consciously or unconsciously used for designing agents -20th century philosophical movements is analytical philosophy. Very simply, analytical philosophy is characterized by the application of a logical method to traditional philosophical problems often using modern formal logic and language analysis2 . After the 1960s such a "pure" logical approach has been questioned and now involves a much more general notion of an "analytic" style, characterized by precision and thoroughness of a narrow topic and opposed to informal and imprecise discussions of broad topics. However, even this approach seems to create debates among analytic philosophers. In a very recent article [START_REF] Crane | Con le migliori 'intenzioni[END_REF] Tim Crane -one of the most important contemporary analytic philosophy philosophers -offers his interesting perspective on the role of analytic philosophy in recent years. In particular in his article he focuses on the study of the mind. Mind in fact has always been the center of interest of analytic philosophy since the work of Bertrand Russell. To study the phenomenon of belief, for example, analytic philosophers looked at the ways in which we talk about beliefs, as well as logic and other properties of our discussion. But there is another reason why analytic philosophers have approached the study of mind and language and it arises from the influence of Wittgenstein on one hand, and by behaviorist psychology on the other.

As we know Wittgenstein argued the impossibility of a "private language", or of a language that was understandable by just one person. If we want to express our thoughts through language, what we express must be publicly accessible in some way: thought in its essence cannot be private. The behaviorist psychology of Skinner, starting from different assumptions, leads to similar conclusions: the only way we can study thought is to look at verbal behavior because, unlike in private thoughts, the behavior can be scientifically verified. The legacy of these perspectives in analytic philosophy is the vision of the mental representation (what Franz Brentano in the nineteenth century called "intentionality") heavily based on the study of language [START_REF] Crane | Con le migliori 'intenzioni[END_REF]. Crane continues asserting that while the approach to mental representation intent -based on language -has given many important results it also has a distorting effect on the correct understanding of the conscious mind.

The German philosopher Edmund Husserl -founder of phenomenology -saw intentionality and consciousness as intrinsically linked: the intent is an intuitive idea, it is how we imagine the world. In addition much in our representation of the world, in perception, thought and emotion, is unconscious. The approach to mental representation based on language is then unable to account adequately for consciousness. While Crane moves towards a doctrine where consciousness is the study of ideas in the private and subjective mind (fundamentally psychology-based) we want to argue for a more extended approach. As we can see, even in analytic philosophy there is a criticism leading towards the concept of subjective mind and uniqueness. While analytic philosophy seems to understand problems arising from the separation of mind and body it does not address the uniqueness problem, nor the body integration in depth. If we consider most of the logical approaches derived from the above mentioned philosophers then it is not surprising that agents have been conceived in the way described before. With regard to the language, for example, what agents designers did was closer to creating a communication language between agents -the explicit part inside thinking -rather than a language for a thinking being.

Perspectives

At the end of this paper we can then say that our provocative title can be explained this way: in the present state of things agents cannot have a sex not only because they have an abstract body -manipulated by an abstract mind -but also because "Universal Man" has no sex. In fact this paper started with the consideration that in the agent research community most current concepts regarding identity originate from the translation of a particular philosophical concept, the "Universal Man". For this reason the paper has shown that a part of contemporary philosophy and agent design has already abandoned the dualism of mind / body. In addition contemporary philosophy has an interesting way of looking at the concept of uniqueness, performance and environment as interlaced and interacting. At the same time this paper has underlined the fact that the approaches based on logic and symbolic representations have the possibility to shift towards more complex approaches. This could be done using more recent philosophical concepts like, for instance, the ones we analyzed in this paper at least from a theoretical point of view. Although the problem solving vision is useful in many ways, integrating different concepts can lead to a more global vision about autonomous agent design.

Obviously identity is only one of the concepts that could be analyzed and the analysis proposed in the core part of this paper makes up only a subset of the concepts that can consist in identity. For example a whole section could be dedicated to the relational self -i.e. the way in which an agent holds a representation of other agents -vs. collective self -i.e. the way a group of agents create their identity. However we believe that even through this brief analysis we can underline interesting paths agent design can follow. Evidently the choice of which kind of concept to use is not neutral and would influence the final system. At the same time we are aware that agent design is an action-oriented field which needs to avoid the trap of "analysis paralysis" and to move towards knowledge scaling. For this reason we suggest replacing a top-down logical approach with a bottom-up enactive approach. Based on our analysis we suggest that agent design can integrate the following concepts.

Uniqueness

The concept of uniqueness could be very interesting to integrate in agent design for example in a mixed environment -involving virtual agents as well as human agents. In this case uniqueness can be used by human agents for connecting with their own virtual ones.

Autopoiesis

The concept of autopoiesis -maintaining organization through the evolution of the structure despite the environmental disturbances -is strictly linked with the uniqueness one. In autopoietic systems uniqueness may be considered as a particular trajectory of the coupling between organization and structure.

Enaction The concept of enaction could be integrated in agent design in order to overcome the dualism of mind / body. Going beyond this dualism can help to create agents which are more adaptive to unknown environments because of the physical grounding of their interactions.

The concepts we suggest integrating in the agent design paradigm are nothing more than necessary steps -but not necessarily sufficient -to reach the autonomy stage. However we strongly believe that as long as the design of agents is mainly based on analytic philosophy, we can only have an enlargement of the domain and not a paradigm shift -i.e. a change in the basic assumptions, or paradigms [START_REF] Kuhn | The Structure of Scientific Revolutions[END_REF] -which is at the basis of science.

Indeed, the genealogy of the concept of "Universal Man" should not be attributed to Mary Wollstonecraft but dates back to the Idea of Plato, Plotinus, and the republican ideals of the universal man so dear to the French Enlightenment.

Some of the most important philosophers of this tradition are Bertrand Russell, Gottlob Frege, Ludwig Wittgenstein, Willard Van Orman Quine, Saul Kripke and David Lewis.


Maxime Hébrard

Béatrice Bocquet

Isabelle Meunier

Delphine Coustes-Chazalette

Emilie Hérald

Audrey Sénéchal

Anne Bolland Augé

Diana Zelenika

Gaël Manès

Christian P Hamel
email: christian.hamel@inserm.fr

Maxime Hebrard

Gaël Manes

Anne Bolland-Augé

Combining gene mapping and phenotype assessment for fast mutation finding in non consanguineous autosomal recessive retinitis pigmentosa

Keywords: retinitis pigmentosa, gene mapping, autosomal recessive inheritance, non consanguineous families, phenotype characterization

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

Photoreceptor degenerations are the leading cause of inherited blindness [START_REF] Hartong | Retinitis pigmentosa[END_REF] . This is partly explained by the extreme genetic heterogeneity of these conditions with 160 genes currently registered in the RetNet database (www.sph.uth.tmc.edu/retnet), reflecting the vast repertoire of genes necessary for photoreceptor function. Concurrently, there is a variety of phenotypes caused by photoreceptor loss, which are classified in many clinical entities depending on the presence or absence of systemic involvement, the severity and course of the disease, the location and shape of retinal lesions and deposits, the involvement of either rod, cone, or both photoreceptors, and the type of electrical responses to light stimuli. The most frequent clinical entity, non syndromic retinitis pigmentosa (RP), is also the most genetically heterogeneous, with 51 disease causing genes being currently known in this condition. From these, 32 cause autosomal recessive (ar) forms of the disease, accounting for 50 to 60 % of all arRP cases [START_REF] Hartong | Retinitis pigmentosa[END_REF] .

Molecular diagnosis in arRP thus requires the systematic sequencing of 506 exons to cover the 32 genes. This is a tremendous task with conventional sequencing methods, which in addition can miss mutations located in non coding regions. Exome sequencing using high throughput sequencing technologies are powerful new methods, but they still remain costly.

Alternatively, homozygosity mapping in inbred, multiplex families or isolated cases, is time saving by readily pointing at only one or a few regions containing an already known disease causing gene or at new genes/loci [START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF] . This strategy has also been successful in a variable proportion of cases from outbred families which carry a homozygous mutation, due to inbreeding encountered in some populations [START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hildebrandt | A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations[END_REF][START_REF] Harville | Identification of 11 novel mutations in eight BBS genes by high-resolution homozygosity mapping[END_REF] . However, homozygous regions unlinked to disease phenotypes are common in the human genome [START_REF] Li | Long contiguous stretches of homozygosity in the human genome[END_REF] , and therefore may erroneously suggest false candidate regions. In addition, the majority of families originating from countries with mixed populations are outbred and the affected patients carry compound heterozygous mutations. For these families, homozygosity mapping will thus remain uninformative.

Here, we devised a strategy based on gene mapping in non consanguineous families to search for mutations in known genes. In two families, we performed a genome wide SNP analysis and found many candidate chromosomal regions. By determination of a score from genotyping results and by assessment of phenotype features, we selected one candidate gene in each family. This process allowed to identify novel mutations in TULP1 and in the recently described C2ORF71, thus evidencing the potential interest of this method for mutation finding in small outbred families.

METHODS

Patients and clinical investigations

Two non-consanguineous French families (RP127 and RP1459) with non syndromic RP and evidence of autosomal recessive inheritance were recruited (Figure 1A,C). In RP127, two out of six siblings were affected; parents and offsprings from the two affected patients were normal. In RP1459, two out of three sisters were affected; parents were normal. Informed consent and blood samples were obtained from family members. The investigators followed the tenets of the Declaration of Helsinki.

Patients had standard ophthalmologic examination (refractometry, visual acuity, slit-lamp examination, applanation tonometry, funduscopy). Kinetic visual fields were determined with a Goldman perimeter with targets V 4e , III 4e and I 4e . OCT measurement of the macula was performed using an OCT-3 system (Stratus model 3000, Carl Zeiss Meditec, CA) with the software version 3.0. Autofluorescence measurements were obtained with the HRA2 Heidelberg retinal confocal angiograph (Heidelberg Engineering, Dossenheim, Germany) and fundus pictures were taken. Full-fields ERG was recorded using a Ganzfeld apparatus (Metrovision, Pérenchies, France) with a bipolar contact lens electrode on maximally dilated pupils according to the ISCEV protocol [START_REF] Marmor | Standard for clinical electroretinography (2004 update)[END_REF] .

Genotyping and mapping

Genomic DNA was extracted from 10-ml peripheral blood samples by a standard salting out procedure [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] . Eleven members of the RP127 family and the 3 sisters of the RP1459 family were genotyped for 262,270 SNPs (GeneChip Mapping 250K Nsp Array, Affymetrix, Santa Clara, CA) at the Centre National de Génotypage (http://www.cng.fr), Evry, France or at DNAVision, Charleroi, Belgium. Results were analyzed using TASE (for transmitted allele search engine), a software designed in our laboratory (http://www.inmfrancetools.com/TASE) to search for common genotypes in all affected individuals (CGAA test). This test compares every SNP between each individual in the family, and assigns 1 of 3 possible states to each SNP: i) excluding SNP (affected individuals have different genotypes), ii) neutral SNP (all affected individuals share the same genotype with some non affected individuals) and iii) qualifying SNP (all affected individuals share the same genotype while non affected individuals carry another genotype). Candidate chromosomal regions were then defined as stretches of neutral and/or qualifying SNPs encompassing regions that were > 5 Mb and assigned centromeric and telomeric boundaries defined by 2 consecutive excluding SNPs. For each chromosome, the results were displayed as a chart showing the status of each SNP (see http://www.inmfrancetools.com/TASE), and the candidate regions were then compared to the position of known genes and loci for retinal inherited diseases according to the RetNet database (www.sph.uth.tmc.edu/retnet).

Selection of candidate genes

This was based on data issued from gene mapping and phenotype characterization. For a given candidate region, the probability to contain the causative gene usually increases with its coverage (average number of SNPs per megabase) and the qualifying rate (percentage of qualifying SNPs over the total number of SNPs in the region). We calculated a qualifying score defined as the product of coverage*qualifying rate/10 that took into account these two parameters. Regions with the highest qualifying scores were considered as those with the highest probability to contain the causative gene. In parallel, known retinal disease genes present in the candidate regions were listed, and the phenotypes usually caused by mutations in these genes were compared with the phenotype observed in the patients for each family.

For each gene, the comparison was qualified as "concordant" when the candidate genes were indeed responsible for an arRP form similar to that observed in the families or as "non concordant" when candidate genes were responsible for RP (or non RP) phenotypes different from that observed in the family. Only a few occurrences of concordant phenotypes with the highest qualifying scores remained allowing for the selection of a few candidate genes to sequence.

Mutation screening

All exons and exon-intron boundaries of TULP1 (GenBank accession # NM_003322.3) and C2ORF71 (GeneBank accession # NM_001029883.1) were sequenced. Primer pairs chosen for the 15 TULP1 exons and the 2 C2ORF71 exons are available on request. Each PCR was performed in a 25-µl reaction mix containing 50 ng genomic DNA, 2 mM MgCl 2 , 200 µM dNTPs, 0.2 µM of each primers and 1U of Taq DNA Polymerase AmpliTaq Gold (Applied Biosystems, Foster City, CA) in its appropriate buffer. Following the first denaturation at 95°C for 9 min, amplification was carried out for 35 cycles at 95°C for 30 s, at the melting temperature (T m ) of the primers (56°C-60°C) for 1 min and at 72 °C for 1 min, ending with a final extension step at 72 °C for 10 min. PCR products were purified with ExoSap-it clean up (Amersham Biosciences, Piscataway, NJ) and sequenced using the BigDye Terminator cycle sequencing ready reaction kit V3.1 on an Applied Biosystems 3130xL genetic analyser (both Applied Biosystems, Forster city, CA) following manufacturer's instructions. Sequencing results were analyzed by alignment with the Clustalw program (version 1.83).

RESULTS

Clinical description

Family RP127

Proband (II.5) had night blindness and visual field defects since early childhood. At time of presentation (age 42), she had moderate myopia (-1.25(-1.50;40°) OD; -1.00(-1.75;10°) OS).

She was counting fingers on the right eye and had hand motion on the left eye. She had posterior subcapsular cataract and intraocular pressure was normal at 14 mmHg on both eyes.

Her fundus showed a bilateral macular atrophy and dense bone spicule-shaped pigment deposits in retinal periphery (Figure 2A,B). Retinal vessels were highly attenuated and optic discs were pale. She was seen again at age 55 and had only light perception in both eyes.

Visual field was undetectable.

Her affected eldest brother (II.2) also had night blindness and visual field defects since early childhood, leading to the diagnosis of RP at age 5. He could never drive. Yet, he was able to read until the age of 33, at which time he had cataract surgery but no improvement in visual acuity. At 40, he was virtually blind and needed a white stick to move outside. At time of presentation (age 54) he had no light perception in the right eye and faint light perception in the left eye. Intraocular pressure was normal at 14 mmHg in both eyes. The macula had a yellowish, disorganized aspect and many bone spicule-shaped pigment deposits were present in mid periphery (Figure 2C,D). Retinal vessels were hardly visible and he had waxy optic discs. He died from repeated cardiac infarctions. Mother (I.2), patients' sibs (II.1, II.3, II.4, II.6) and patients' children (III.1, III.2, III.3, III.4) were examined; no signs of RP were detected. We concluded that patients II.2 and II.5 had severe arRP.

Family RP1459

Proband (II.2) had moderate dyschromatopsia since early childhood. Later on, at age 14 she noticed moderate night blindness and photophobia. She had difficulties to distinguish fine objects in far sight but had no reading difficulties. She did not complain of loss in the peripheral visual field and was able to drive her car in day time. At the time of presentation (age 25), her visual acuity was moderately decreased at 20/30 on both eyes with myopic refraction -6.75(-1.75;45°) OD; -5.75(-0.75;110°) OS. Intraocular pressure was normal at 14 mmHg on both eyes. Lenses were transparent. Her fundus showed slightly discolored foveas with loss of foveal reflex, slightly pale optic discs and vascular attenuation (Figure 3A). There were no pigment deposits or retinal atrophy. Autofluorescence of macula was moderately heterogeneous whereas that of peripheral retina was normal (Figure 3B). OCT scans showed thinning of the retina with loss of the IS/OS line in both fovea and macula (Figure 3E).

Goldman perimetry showed that peripheral isopter V4e was normal (90° temporal, 60° nasal) but there was a relative central scotoma on 20° around fixation at I4e in both eyes. Moderate tritanopia was confirmed on desaturated 15 HUE test. ERG testing showed barely detectable responses both in scotopic (responses only at maximal stimulation) and photopic conditions (responses only at 30-Hz flickers) (Figure 3G).

Affected sister (II.1) had moderate dyschromatopsia and night blindness since early childhood. At age 20 she noticed moderate photophobia. She had slight reading difficulties. She did not complain of loss in the peripheral visual field and was able to drive her car both in day and night time. At the time of presentation (age 30), her visual acuity was moderately decreased at 20/40 on both eyes with +1.50(-1.75;95°) OD; +1.50(-1.75;70°) OS. Lenses were transparent. Her fundus showed slightly discolored foveas with loss of foveal reflex, normal optic discs and moderate vascular attenuation (Figure 3C). There were no pigment deposits or retinal atrophy. Autofluorescence of macula was moderately heterogeneous whereas that of peripheral retina was normal (Figure 3D). OCT scans showed thinning of the retina with loss of the IS/OS line in the macula and relative sparing in the fovea (Figure 3F). Goldman perimetry showed that peripheral isopter V4e was moderately decreased (70° temporal, 50° nasal) with relative central scotoma on 20° around fixation at I4e in both eyes. There was non systematized dyschromatopsia confirmed on desaturated 15 HUE test. ERG testing showed barely detectable responses in photopic conditions but responses in scotopic conditions were still present even for the lowest stimuli, although attenuated (Figure 3G).

The parents (II.1, II.2) and sister II.3 had no symptoms, normal visual acuity and funduscopy, indicating that they did not have RP. Yet, they all had slightly decreased photopic responses at ERG (Figure 3G). Based on dyschromatopsia, decreased visual acuity while peripheral visual field was normal, and ERG finding in II.1, we concluded that affected sisters II.1 and II.2 had the cone rod dystrophy form of arRP, with some intrafamilial variations.

Gene mapping, selection of candidate genes and mutation identification

Family RP127

the TASE software, we found that 33 chromosomal regions were common to the two affected patients, with an average length of 21 Mb (range: 5-68 Mb) and coverage of 75.5 SNPs/Mb (range: 0.6-133 SNPs/Mb) (Table 1). The qualifying rate (QR) and qualifying score (QS) were first calculated for the 6 members of the sibship (generation II). The 5 best QS (range: 233.1-56.5) were found to correspond (from highest to lowest QS) to chromosomes 3, 18, 6, 11 and 5. Adding healthy members of the family from generations I and III should decrease the probability that affected sibs had common genotypes with another member of the family only by chance. When the mother I.2 (generation I) and the 4 members of generation III were added (11 members), the value of the QS therefore dropped but highlighted the genotype specific to the affected individuals. The order of the 5 best QS (range 20.4-9.5) changed to the chromosome 11 region first (QS 20.4), followed by chromosome 6 (QS 14.1) and then 1, 3 and 5. We then listed all possible genes and phenotypes for each chromosomal region and compared them with the phenotype of affected members of family RP127. Fifteen of the 33 regions contained 31 candidate genes previously reported in inherited retinal diseases (Table 1). When comparing phenotypes caused by these candidate genes with the severe non syndromic arRP of the two affected family members, it was found that RPE65, ABCA4, SNRNP200, RHO, PROM1, TULP1, IMPDH1, RGR, SPATA7, TTC8, CNGB1 and CA4 were concordant. Of these, only TULP1 was in the chromosomal regions with the 5 best QS. Since TULP1 was repeatedly reported in severe RP cases and was also the candidate gene of chromosome 6 region with the second highest QS, we sequenced all TULP1 coding exons and exon-intron boundaries in the 11 family members. We found two nucleotide changes, c.932G>A in exon 10 and c.1025G>A in exon 11, resulting in two novel amino acid substitutions, p.Arg311Gln and p.Arg342Gln, respectively (Figure 1B). These substitutions segregated with the RP phenotype (Figure 1A). Indeed, affected patients II.2 and II.5 carried both mutations in trans whereas healthy individuals carried either only one mutation or wildtype alleles. These changes were not found in 57 unrelated controls, indicating that they were likely to be disease causing mutations.

Family RP1459

To test whether this strategy would be efficient in a smaller sibship, we performed genome wide SNP genotyping on the 3 sisters of family RP1459. Using the TASE software, we found that 40 chromosomal regions were common to the two affected sisters, with an average length of 19 Mb (range: 5-87 Mb) and coverage of 78 SNPs/Mb (range: 1-129 SNPs/Mb) (Table 2).

The 7 best QS (range: 461.7-369.7) were found to correspond (from highest to lowest QS) to regions of chromosomes 6, 5, 2, 1, 8, 6 and 2. Eight of the 40 chromosomal regions contained 14 candidate genes previously reported in inherited retinal diseases. When comparing phenotypes caused by these candidate genes with the arRP of the two affected sisters, it was found that 4 genes, C2ORF71, ZNF513, PRCD and PDE6G were concordant. Of these, only C2ORF71 and ZNF513 were included in one of the 7 chromosomal regions with the best QS, namely the chromosome 2 region being the seventh in QS rank. Among these two genes, C2ORF71 had previously been described in cone rod dystrophies [START_REF] Collin | Mutations in C2ORF71 cause autosomalrecessive retinitis pigmentosa[END_REF][START_REF] Nishimura | Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71[END_REF][START_REF] Sergouniotis | A survey of DNA variation of C2ORF71 in probands with progressive autosomal recessive retinal degeneration and controls[END_REF] , a phenotype corresponding closely to what was found in the two sisters of family RP1459. The exons of C2ORF71 were therefore sequenced in the 5 family members. We found two variants in exon 1; an insertion, c.2327_2328insC, and a deletion, c.2328_2344del17, both resulting in frameshifts, p.Leu777PhefsX34 and p.Leu777AsnfsX28, respectively (Figure 1D). These variants segregated with the RP phenotype (Figure 1C). Indeed, the two affected sisters carried both variants in trans whereas healthy sister and parents carried only one variant.

These variants were therefore likely to be disease causing mutations.

DISCUSSION

With the advent of clinical trials for inherited retinal dystrophies, it is required to identify the causative gene. Indeed, the molecular identification permits the diagnosis of the RP subtype, a better patient follow up and prediction of disease course. This will also be necessary for gene therapy. However, molecular diagnosis in arRP, the most genetically heterogeneous form of inherited retinal disease, currently requires the screening of 32 genes, a process which was never fully completed by any research group because it is time and money consuming. To circumvent this difficulty, various genetic tests have been developed based on use of microchips testing for known mutations (Asper Biotech, Tartu, Estonia), use of re-sequencing microchips [START_REF] Mandal | Sequencing arrays for screening multiple genes associated with early-onset human retinal degenerations on a high-throughput platform[END_REF] or preferential sequencing of mutation hot spots [START_REF] Karra | Population haplotypes of exon ORF15 of the retinitis pigmentosa GTPase regulator gene in Germany : implications for screening for inherited retinal disorders[END_REF] . Yet, these strategies miss unknown mutations and/or rare genes. Another possibility is to perform phenotype-genotype correlations to orient genetic testing towards one or a few genes. Although this could be very efficient in rare occasions in which a particular clinical feature is specific to a single gene, like para-arteriolar preservation of the retinal pigment epithelium in CRB1 mutations [START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF] , in most cases the RP phenotype simply belongs to a broad class of RP subtype, such as severe versus moderate, or rod-cone versus cone-rod dystrophy, all of which contain many genes to test.

Homozygosity mapping proved to be a very efficient method for identification of mutations and gene discovery in small inbred RP families [START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Littink | Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations[END_REF] as well as in about one third of patients from outbred families who carry homozygous mutations [START_REF] Hildebrandt | A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations[END_REF][START_REF] Harville | Identification of 11 novel mutations in eight BBS genes by high-resolution homozygosity mapping[END_REF][START_REF] Littink | Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations[END_REF] . However, there remains about two thirds of outbred families, being the most frequent in countries with highly mixed populations, in which affected patients carry compound heterozygous mutations. In these families, gene mapping could lead to the causative gene if there is a sufficient number of affected patients in the sibship, a rather rare occurrence today [START_REF] Bergmann | Mutations in the gene encoding the Wntsignaling component R-spondin 4 (RSPO4) cause autosomal recessive anonychia[END_REF] . Therefore, gene mapping of non consanguineous families is usually used to ascertain a locus previously identified by homozygosity mapping, and to increase the probability of finding mutations in a novel gene.

We show in this study that combining gene mapping and phenotype-genotype correlation in small outbred families leads to the identification of the causative genes among several dozen of theoretically possible RP genes. Gene mapping is based on the assumption that the responsible gene must be present in chromosomal regions where affected patients from a single sibship have the same genotype. Although we found that in 260,000-SNP microchips many regions respond to this criterion, we could restrain the search to a few regions which had the highest qualifying scores (QSs). The QS varies for the one part with the SNP coverage of the chromosomal region, and for the other part with the density of qualifying SNPs in the region, herein called the qualifying rate (QR). Indeed, if the QR is low, this means that most SNPs of a given region have a genotype common to affected patients and to unaffected individuals of the kindred, making it unlikely to contain the causative gene.

Conversely, if the QR is high, genotype of affected patients is different from that of unaffected individuals for many SNPs, therefore increasing the probability for the given region to contain the causative gene. With the increasing number of genes described in inherited retinal diseases, there are also an increasing number of precise phenotype descriptions [START_REF] Sparrow | The retinal pigment epithelium in health and disease[END_REF] . Thus, there were only a limited number of genes that fulfilled the criteria of a high QS and phenotype concordance. Therefore, combining both approaches appears useful for rapid mutation identification.

The tubby-like protein 1 (TULP1) contains 542 amino acids, among which the ~260 Cterminal amino acids form the tubby domain conserved in Tubby and in the three TULP proteins (Figure 4A). TULP1 is specific to photoreceptor cells and is expressed in the inner segment, connecting cilium and synapses of photoreceptors. Today, 24 pathogenic variants have been described in TULP1, including 12 missense and 12 nonsense or frameshift mutations [START_REF] Gu | Tubby-like protein-1 mutations in autosomal recessive retinitis pigmentosa[END_REF][START_REF] Hagstrom | Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa[END_REF][START_REF] North | Molecular characterization of TUB, TULP1, and TULP2, members of the novel tubby gene family and their possible relation to ocular diseases[END_REF][START_REF] Hanein | Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis[END_REF][START_REF] Banerjee | TULP1 mutation in two extended Dominican kindreds with autosomal recessive retinitis pigmentosa[END_REF][START_REF] Hollander | Novel compound heterozygous TULP1 mutations in a family with severe earlyonset retinitis pigmentosa[END_REF][START_REF] Kondo | A homozygosity-based search for mutations in patients with autosomal recessive retinitis pigmentosa, using microsatellite markers[END_REF][START_REF] Mataftsi | Novel TULP1 mutation causing leber congenital amaurosis or early onset retinal degeneration[END_REF][START_REF] Paloma | Novel mutations in the TULP1 gene causing autosomal recessive retinitis pigmentosa[END_REF][START_REF] Lewis | Tubby-like protein 1 homozygous splice-site mutation causes early-onset severe retinal degeneration[END_REF][START_REF] Abbasi | A novel splice-site mutation of TULP1 underlies severe early-onset retinitis pigmentosa in a consanguineous Israeli Muslim Arab family[END_REF] . Ten out of the 12 missense mutations are present in the tubby domain. The two novel missense mutations R311Q and R342Q that we have found in this study also affect amino acids of the tubby domain. Using a protein data bank software (http://www.rcsb.org/pdb), we predict that Q311 would no longer be able to interact with F535 as does wild type R311 do, therefore possibly destabilizing the protein. We also speculate that Q342, in contrast to wild type R342, could increase the flexibility of the external loop of TULP1, therefore preventing protein-protein interactions. The early onset severe RP observed in the two patients carrying these mutations was in accordance with the phenotype described in TULP1 patients. Thus, R311Q and R342Q are likely pathogenic changes.

C2ORF71 encodes a 1288-amino acid protein with no known homology. The mRNA was shown to be specifically expressed in photoreceptors [START_REF] Collin | Mutations in C2ORF71 cause autosomalrecessive retinitis pigmentosa[END_REF][START_REF] Nishimura | Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71[END_REF] . In these cells, C2ORF71 could possibly be associated with the photoreceptor connecting cilium and also play a role in the photoreceptor development since it associates with basal bodies of the developing cilium [START_REF] Nishimura | Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71[END_REF] .

Using homozygosity mapping of consanguineous families, five pathogenic variants were recently reported in this gene [START_REF] Collin | Mutations in C2ORF71 cause autosomalrecessive retinitis pigmentosa[END_REF][START_REF] Nishimura | Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71[END_REF] , four of them being nonsense or frameshift mutations. A medium scale systematic screening in 191 arRP and 95 Leber congenital amaurosis patients did not find any mutation, suggesting that C2ORF71 is not a frequent gene of inherited retinal dystrophies [START_REF] Sergouniotis | A survey of DNA variation of C2ORF71 in probands with progressive autosomal recessive retinal degeneration and controls[END_REF] . The two novel variants found in this study are also frameshift mutations in compound heterozygous patients, unambiguously indicating that they are pathogenic. Among the five previously reported families, one Dutch family was described in more details, featuring criteria of cone rod dystrophy based on worse cone ERG responses when compared to rod ERG responses. This corresponds well to patient II.1 from this study, showing that phenotype genotype correlation was relevant and, together with gene mapping, led to efficient mutation finding.

In conclusion, a combined approach of gene mapping and phenotypic assessment was efficient to find, among many possible chromosomal regions and causative genes, the mutations in two non consanguineous families.

TITLES AND LEGENDS TO FIGURES

Figure 1 :

 1 Figure 1: Pedigrees and sequence analysis in 2 families segregating autosomal recessive retinitis pigmentosa. A, C: pedigree of families RP127 (A) and RP1459 (C); blackened symbols are affected individuals, mutation genotypes for TULP1 (A) or C2ORF71 (C) is indicated under each family member ("+" means a wild-type allele). B, D: electropherograms; the normal sequence is written in black italic and the mutated nucleotides are in red. B, TULP1 sequence for each of the two mutations in patient (indicated above) compared to wild type (normal) is shown. D, C2ORF71 sequence for each of the two mutations in either father or mother (indicated above) is shown and compared to that of patient who carry both mutations in exon 1 and of wild type individual (normal).

Figure 2 :

 2 Figure 2: Fundus images of affected patients in family RP127. A, right and B, left eye posterior poles of patient II.5 at age 42 showing round shape atrophy of the macula, advanced atrophy of the peripheral retina, pigment deposits and narrowing of retinal vessels. C, superior and D, inferior retina in the right eye of patient II.2 at age 54 showing major atrophy of the whole retina including retinal periphery and macula, with pigment deposits, tenuous retinal vessels and waxy pale optic discs.

Figure 3 :

 3 Figure 3: Clinical findings in family RP1459. A, C, fundus photographs and B, D, fundus autofluorescence of left eye of patients II.2 (A, B) and II.1 (C, D) showing slight discoloration of fovea, loss of normal foveal reflex and moderate narrowing of retinal vessels (A, C) and alteration of the foveal retinal pigment epithelium (B, D). E, F, OCT scans of left eye of patients II.2 (E) and II.1 (F) showing thinning of the retina with relative preservation of foveal photoreceptors. G, electroretinogram recordings showing the dramatic reduction of light-

Table 1 : data analysis for family RP127

 1 Length is in Mb; coverage is the average number of SNPs/Mb; QR: qualifying rate is the percentage of qualifying SNPs/total number of SNPs; QS: qualifying score is the product of coverage*QR/10; BBS: Bardet Biedl syndrome; CD: cone dystrophy; CRD: cone rod dystrophy; FEVR: familial exudative vitreoretinopathy; LCA: Leber congenital amaurosis;

				6 members	11 members			Concordance with phenotype of
	Chrom Length Coverage	QR	QS	QR	QS	Genes	Disease	affected patients
	1	7	52	4,87	25,3	2,5	13,0
	1	51	95	0,12	1,1	0	0,0	RPE65	LCA, RP	Concordant
								ABCA4	MD, CRD, RP	Concordant
								COL11A1	Stickler	Non concordant
								GNAT2	Achromatopsia	Non concordant
	1	24	1	11,11	1,1	0	0,0
	2	12	102	0,08	0,8	0	0,0
	2	18	110	2,5	27,5	0	0,0	EFEMP1	MCDR	Non concordant
	2	5	0	0	0,0	0	0,0	SNRNP200	RP	Concordant
	3	26	93	1,2	11,2	0,12	1,1
	3	5	113	20,63	233,1	0,88	9,9
	3	60	84	0,05	0,4	0,02	0,2	IQCB1	SLS	Non concordant
								RHO	RP	Concordant
								NPHP3	SLS	Non concordant
								CLRN1	USH	Non concordant
	4	9	120	1,8	21,6	0	0,0	PROM1	RP, MCDR	Concordant
	5	16	90	6,28	56,5	0,71	6,4
	5	27	98	3,21	31,5	0,97	9,5
	6	32	99	8,12	80,4	1,42	14,1	TULP1	LCA, RP	Concordant
	6	20	106	0,09	1,0	0,09	1,0
	7	51	78	0,07	0,5	0	0,0	TSPAN12	FEVR	Non concordant
								IMPDH1	RP	Concordant
								OPN1SW	Tritanopia	Non concordant
	8	6	18	0	0,0	0	0,0
	9	29	0	0	0,0	0	0,0
	10	5	126	0,15	1,9	0,16	2,0
	10	68	96	3,9	37,4	0,37	3,6	CDH23	USH	Non concordant
								CDHR1	CRD	Non concordant
								RGR	RP	Concordant
								PDE6C	CD	Non concordant
								RBP4	RPE dystrophy	Non concordant
								PAX2	Coloboma	Non concordant
	11	21	117	0,16	1,9	0	0,0	USH1C	USH	Non concordant
	11	18	63	0,26	1,6	0,18	1,1
	11	14	88	7,32	64,4	2,32	20,4	MYO7A	USH	Non concordant
	11	14	88	0,24	2,1	0,08	0,7	C1QTNF5	MCDR	Non concordant
	14	19	77	2,99	23,0	0,41	3,2	SPATA7	LCA, RP	Concordant
								TTC8	BBS, RP	Concordant
	16	6	0	0	0,0	0	0,0
	16	9	97	5,79	56,2	0,22	2,1	RPGRIP1L	JBTS	Non concordant
								BBS2	BBS	Non concordant
								CNGB1	RP	Concordant
	17	16	50	0	0,0	0	0,0	UNC119	CRD	Non concordant
	17	22	58	0	0,0	0	0,0	CA4	RP	Concordant
	18	35	91	17,08	155,4	0,64	5,8
	18	11	133	0,13	1,7	0	0,0
	19	8	1	0	0,0	0	0,0
	21	14	93	5,78	53,8	0,3	2,8
	22	5	47	3,36	15,8	0	0,0

MD: macular dystrophy; RP: retinitis pigmentosa; SLS: senior loken syndrome

Table 2 : data analysis for family RP1459

 2 Length is in Mb; coverage is the average number of SNPs/Mb; QR: qualifying rate is the percentage of qualifying SNPs/total number of SNPs; QS: qualifying score is the product of coverage

								Concordance
								with phenotype
								of affected
	Chrom Length	Coverage	QR	QS	Genes	Disease	patients
	1	12	118	34.97	412.6
	1	24	2	11.26	2.2
	1	16	89	38.93	346.4	SDCCAG8	Nephronophtisis	Non concordant
	2	25	98	37.73	369.7	C2ORF71	RP, CRD	Concordant
						ZNF513	RP	Concordant
	2	8	90	25.79	232.1
	2	7	4	26.47	10.5
	2	6	95	0	0
	2	14	88	24.08	211.9
	2	9	106	39.54	419.1
	3	5	6	15.62	9.3
	4	6	116	28.98	336.1
	4	21	101	35.18	355.3
	4	44	91	3.33	30.3
	5	5	22	2.72	5.9
	5	10	84	39.42	331.1	VCAN	Wagner	Non concordant
	5	7	128	34.29	438.9
	6	11	101	29.95	302.5
	6	48	95	40.58	385.5	ELOVL4	MD	Non concordant
	6	7	114	40.5	461.7
	7	13	129	25.97	335
	7	6	69	27.64	190.7
	8	5	105	21.17	222.2
	8	6	15	0	0
	8	31	101	39.96	403.5
	9	31	1	34.21	3.42
	10	40	92	13.06	120.1
	10	18	90	23.14	208.2	CDH23	Usher syndrome	Non concordant
	10	16	99	35.73	353.7	OAT	Gyrate atrophy	Non concordant
	11	17	99	6.74	66.7
	12	87	96	33.02	316.9	BBS10	BBS	Non concordant
						CEP290	LCA, SLS, JS	Non concordant
						COL2A1	Sticker, Wagner	Non concordant
						RDH5	FA, CD	Non concordant
	12	20	75	3.58	26.8
	13	11	114	32.4	369.3
	15	20	95	0.26	2.47
	16	11	106	28.4	301
	16	13	5	11.94	5.9
	17	13	42	21.06	88.4	USH1G	Usher syndrome	Non concordant
						PRCD	RP	Concordant
						PDE6G	RP	Concordant
	18	32	89	27.6	245.6
	18	10	119	6.85	81.5
	19	9	12	24.32	29.1
	19	8	55	2.02	11.1

*QR/10; BBS: Bardet Biedl syndrome; CD: cone dystrophy; CRD: cone rod dystrophy; FA: fundus albipunctatus; JS: Joubert syndrome; LCA: Leber congenital amaurosis; MD: macular dystrophy; RP: retinitis pigmentosa; SLS: senior loken syndrome; Stickler: Stickler syndrome; Wagner: Wagner disease

Acknowledgments: we thank the patients and their family.

Conflict of interest: NONE

Funding: this work was supported by private foundations (Fondation des Aveugles et Handicapés Visuels de France, Formicoeur, Information Recherche sur la Rétinite Pigmentaire, Retina France, SOS Rétinite and UNADEV), Centre National de Génotypage. INSERM and UNADEV support the fellowship for MH.


VUMC C W Ang

D W Notermans

A M Simoons-Smit

T Herremans

Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISA's and five immunoblots

Keywords: Lyme, immunoblot, neuroborreliosis, recombinant antigens, diagnostic test

We investigated the influence of assay choice on the results in a two-tier testing algorithm for detection of anti-Borrelia antibodies. 89 serum samples from clinically well-defined patients were tested in eight different ELISA systems based on whole cell antigens, whole cell antigens supplemented with VlsE and assays using exclusively recombinant proteins. A subset of samples was tested in five immunoblots: one whole cell blot, one whole cell blot supplemented with VlsE and three recombinant blots. The number of IgM and/or IgG positive ELISA results in the group of patients suspected of Borrelia infection ranged from 34% to 59%. The percentage of positives in cross-reactivity controls ranged from 0-38%.

Comparison of immunoblots yielded large differences in inter-test agreement and showed at best a moderate agreement between tests. Remarkably, some immunoblots gave positive results in samples that had been tested negative by all eight ELISA"s. The percentage of positive blots following a positive ELISA result depended heavily on the choice of ELISAimmunoblot combination. We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity. The choice of ELISA-immunoblot combination severely influences the number of positive results, making exchange of testresults between laboratories with different methodologies hazardous.

Introduction

Lyme disease is caused by Borrelia spp. In Europe, infection is mostly caused by B. afzelii and B. garinii, while in the United States, Borrelia burgdorferi sensu stricto is the causative agent [START_REF] Stanek | Lyme borreliosis: Clinical case definitions for diagnosis and management in Europe[END_REF]. Lyme disease manifests in a myriad of clinical ways, including erythema migrans, arthritis, carditis and neuroborreliosis [START_REF] Stanek | Lyme borreliosis: Clinical case definitions for diagnosis and management in Europe[END_REF]. Extracutaneous Lyme disease requires laboratory confirmation by culture, PCR or antibody determination [START_REF] Wilske | MiQ-12 Lyme-Borreliosis (English internet version)[END_REF][START_REF] Brouqui | Guidelines for the diagnosis of tick-borne bacterial diseases in Europe[END_REF]. Culture is only available in a limited number of laboratories, and the value of PCR in the diagnosis of various forms of Lyme disease is of limited use [START_REF] Wilske | MiQ-12 Lyme-Borreliosis (English internet version)[END_REF][START_REF] Brouqui | Guidelines for the diagnosis of tick-borne bacterial diseases in Europe[END_REF]. Therefore, serological assays are the main method used to diagnose extracutaneous forms of Lyme disease.

Current guidelines for the diagnosis of Lyme disease include a two-tier testing algorithm [START_REF] Wilske | MiQ-12 Lyme-Borreliosis (English internet version)[END_REF][START_REF] Brouqui | Guidelines for the diagnosis of tick-borne bacterial diseases in Europe[END_REF]. First an ELISA is performed, followed by confirmation of positive ELISA results with an immunoblot. This two-step procedure was initiated because first generation ELISA"s for the detection of anti-Borrelia antibodies lacked specificity. The inclusion of a second, more specific, serological method made it possible to exclude false positive ELISA samples [START_REF] Wilske | MiQ-12 Lyme-Borreliosis (English internet version)[END_REF][START_REF] Wilske | Microbiological and serological diagnosis of Lyme borreliosis[END_REF].

Many diagnostic assays are currently commercially available, and manufacturers have developed them to increase their sensitivity and specificity. During the last decade, assays using a peptide from the sixth invariant region (C6) of the variable major protein-like sequence-expressed (VlsE) of B. burgdorferi have been shown to be promising [START_REF] Liang | Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE[END_REF][START_REF] Steere | Prospective study of serologic tests for Lyme disease[END_REF].

Laboratories can choose between ELISA"s and immunoblots using sonicated whole cell antigens, whole cell antigens combined with recombinant antigens (VlsE C6 peptide) and exclusively recombinant antigens. Due to this array of serological tests, there are an almost indefinite number of possible combinations between ELISA and immunoblot in a two-tier testing scheme. Comparing anti-Borrelia test results between laboratories and studies may be impossible if tests with widely diverging sensitivities and specificities are used [START_REF] Bakken | Interlaboratory comparison of test results for detection of Lyme disease by 516 participants in the Wisconsin State Laboratory[END_REF].

The aim of the present study was to compare a wide range of ELISA assays and immunoblots, based on either whole cell or recombinant antigens for detecting anti-Borrelia antibodies. We also aimed to investigate the influence of assay choice on results in a two-tier testing algorithm (ELISA followed by immunoblot). Therefore, we tested serum samples in eight ELISA systems and five immunoblots, covering the entire spectrum of native and recombinant antigens.

Patients and methods

Patients

Serum samples were selected from 89 clinically well-defined individuals. Fifty-nine samples were from patients suspected of Borrelia infection (skin manifestations, n = 8; neurological symptoms, n = 26; arthritic symptoms, n = 11; ocular symptoms, n =4; other, n = 10).

Fourteen samples were from healthy controls, and 16 came from patients with a high possibility for cross-reacting antibodies (syphilis patients, n = 10; Mycoplasma pneumoniainfected patients based on symptoms consistent with M. pneumoniae infection and a positive result for anti-M. pneumoniae IgM and IgG with a Virion/Serion ELISA , n = 6).

Methods

Serum samples were tested in eight different ELISA systems. Three assays were based on sonicated whole cell antigens (Diacheck/Moran anti-Borrelia, VIDAS, and Virion/Serion ELISA Classic Borrelia burgdorferi), three assays with sonicate whole cell antigens supplemented with VlsE for IgG anti-Borrelia antibodies (Dade Behring Enzygnost Lyme link VlsE, Euroimmun Anti-Borrelia plus VlsE ELISA, and Genzyme Virotech Borrelia afzelii + VlsE ELISA) and two assays using recombinant proteins (Immunetics C6 Lyme ELISA Kit, and Mikrogen recomWell Borrelia). A subset of samples from 31 patients suspected of Borrelia infection were also tested in five different immunoblots. This group consisted of the following patients: skin manifestations, n = 3; neurological symptoms, n = 15; arthritic symptoms, n = 6; ocular symptoms, n =2; other, n = 5. One whole cell blot (homemade using B. afzelii strain A39 cell sonicate, RIVM), one whole cell blot supplemented with VlsE (Viramed Borrelia "MiQ" + VlsE ViraBlot) and three recombinant blots (Euroimmun Euroline-RN-AT, Mikrogen recom Line Borrelia, and Genzyme Virotech Borrelia Europe Line). A total of 31 samples were tested in all immunoblots.

Manufacturer-suggested cut-off levels and interpretation criteria were used for ELISA"s and immunoblots. Statistical analysis was performed using SPSS version 16.0 (SPSS Inc, Chicago, Ill).

Results

As expected, there was considerable discordance between the eight ELISA"s. We tested 89 samples from patients and controls on all eight ELISA"s. Of the complete set of serum samples 35/89 (39%) were negative in all assays, while 16/89 (18%) were positive in all assays. The remaining 38/89 (43%) samples were positive in 1 to 7 ELISA"s.

In the 59 patients that were suspected of Borrelia infection we observed a wide range of positive results, with percentages of positive ELISA"s varying between 34-61% (Table 1). We did not observe a relation between the fraction of positive results and the nature of antigen used for the ELISA. The specificity of the ELISA"s also varied widely. Although we had only small numbers of positive tests in healthy controls, some ELISA"s produced up to 38% of positive tests in the cross-reactivity group (syphilis and Mycoplasma pneumonia-infected patients).

We aggregated results from the IgM and IgG tests and assessed them using a kappa statistic to determine agreement between the ELISA"s. The kappa-values ranged from 0.41 (moderate agreement) to 0.79 (substantial to good agreement), emphasizing the differences between the ELISA"s (Table 2). The choice of antigen does not seem to influence the level of agreement. Even the lowest kappa-values were observed between two "whole cell + VlsE" ELISA"s (0.43).

We tested a subset of 31 serum samples from patients suspected of Borrelia infection in all five immunoblots. Samples were from patients with positive and negative ELISA results, allowing us to investigate the specificity of the immunoblots. In general, we observed much lower agreement for the immunoblots than for the ELISA"s. Kappa values ranged from 0 (poor agreement) to 0.84 (good agreement), indicating that for many samples the outcome of the immunoblot is highly dependent on the choice of manufacturer (Table 3). Inter-blot agreement was disappointingly low for IgM, and much higher for IgG (Table 3). Interestingly, recombinant blots did not have a higher agreement than whole cell blots, and there was limited agreement even between recombinant blots. The highest agreement was for the The influence of assay choice is further illustrated by investigation of the relationship between each ELISA and the fraction of positive blots. Surprisingly, we found anti-Borrelia immunoblot reactivity in samples that were negative in all eight ELISA"s. These are samples that normally would not have been tested in immunoblot. Again, this was not dependent on the nature of the antigen used for the immunoblot. For the Euroimmun immunoblot, 4/11 lack sensitivity, since samples that were positive in six to all eight of the tested ELISA"s remained negative in all immunoblots. Some of these samples were from Lyme disease patients with a short duration of symptoms, confirming that ELISA"s may have a higher sensitivity than immunoblots during the early phase of a Borrelia infection.

For some ELISA x blot combinations, only about half of the ELISA-positive samples could be confirmed by immunoblot (e.g. VIDAS ELISA x Virotech immunoblot, Table 4). The quality of the other ELISA"s was so high that the majority of ELISA-positive samples were confirmed with immunoblot (e.g. Diacheck/Moran and Enzygnost ELISA"s). When taking into account the lack of specificity of a number of the immunoblots, it is clear that the combination of a non-specific ELISA with a non-specific blot will lead to a high fraction of presumably falsepositive test results.

The ELISA test-value is the final factor influencing the fraction of positive confirmatory blots. When using the Immunetics ELISA as a screening tool, it becomes clear that irrespective of the blot method used, samples with an index >4 are almost always blot-positive. These characteristics make it possible to define groups of ELISA-positive serum samples that do not need immunoblot confirmation.

Discussion

We studied the influence of the choice of detection method on the results of Borrelia serology. We found that Borrelia ELISA"s and immunoblots for detecting anti-Borrelia antibodies have widely divergent sensitivity and specificity, and that immunoblots generally show limited agreement. Analysis of a large number of ELISA x immunoblot combinations revealed large differences between various test strategies in a two-tier testing algorithm.

Although we only studied a limited number of serum samples, our extensive approach allowed us to draw several conclusion based on our observations. Theoretically, the use of recombinant antigens should lead to increased specificity and possibly increased sensitivity as well. This doesn"t seem to be true for the currently available ELISA"s and immunoblots for the detection of anti-Borrelia antibodies. We could not find a clear relationship between the fraction of positive tests, the specificity and the nature of the antigen used for the serological tests. ELISA"s using sonicated whole cell antigens can be sensitive and specific, while recombinant ELISA"s may lack specificity. Therefore, manufacturer claims for the superior performance of assays using recombinant antigens for the detection of Borrelia antibodies must be interpreted with caution.

A two-tier testing algorithm for the detection of anti-Borrelia antibodies is recommended world-wide [START_REF] Wilske | MiQ-12 Lyme-Borreliosis (English internet version)[END_REF][START_REF] Brouqui | Guidelines for the diagnosis of tick-borne bacterial diseases in Europe[END_REF][START_REF] Steere | Prospective study of serologic tests for Lyme disease[END_REF]. However, there are several reasons to reappraise the additional value of an immunoblot confirmatory test in a two-tier testing scheme.

First, the lack of specificity of some immunoblots is counterintuitive. The immunoblot is used as a confirmatory test, although it can be argued that it is merely a supplemental test due to the interdependence of ELISA"s and immunoblots [8]. Theoretically, the use of recombinant antigens should allow discrimination between aspecific antibody reactivity, cross-reactive antibodies and true anti-Borrelia antibodies [START_REF] Wilske | Microbiological and serological diagnosis of Lyme borreliosis[END_REF]. The presence of commercially available immunoblots with low specificity diminishes the value of the immunoblot as a confirmatory test [8]. Furthermore, the two-tier testing scheme was originally proposed to overcome the lack of specificity of Borrelia ELISA"s. This study has shown that not all of the newer generation ELISA"s using recombinant Borrelia antigens have improved specificity than older serological assays [9,10].

Second, the low level of agreement between the different immunoblots is very disappointing, especially for IgM. This low level of agreement, even at the individual band level, makes it hard to compare immunoblot results from different manufacturers.

Third, a mismatch between immunoblot and ELISA may occur during the early phase of infection. There are numerous examplesfrom this and other studiesin which patients with early Lyme disease were initially ELISA-positive and blot-negative [11]. In such cases, immunoblot seroconversion can only be documented in a follow-up sample, and sometimes even this option is blocked because antibiotic treatment may interfere with the development of the anti-Borrelia antibody response [12]. This is an example of better sensitivity in the ELISA compared to the immunoblots. Without detailed knowledge about clinical manifestations and illness duration, reporting these cases as "negative" could lead to erroneous conclusions.

Finally, several groups can be discriminated based on the ELISA value [10] -a "high positive" group exhibiting clinical symptoms consistent with a diagnosis of Lyme disease and which can be reported as "positive" without confirmatory testing, a "low positive" group in which confirmatory testing may be helpful, and lastly a negative group that does not require any further investigation. We do not advocate abandoning the use of immunoblots to confirm anti-Borrelia antibodies, but we do think that only a selection of samples needs confirmatory blotting. Furthermore, knowledge about the lower sensitivity of immunoblots compared to some of the ELISA"s is indispensable in interpreting results.

In conclusion, ELISA"s and immunoblots for detecting anti-Borrelia antibodies have widely

  homemade whole cell blot with the Mikrogen recombinant blot. Additional analysis on the individual band level revealed similarly poor agreement, even in immunoblots containing recombinant antigens. When performing eight different ELISA"s and five different blots, there are 40 possible ELISA x blot combinations. Thirty-one samples were tested in all 40 combinations. A score of 0 indicates a negative result in all ELISA"s and all blots, while a score of 40 indicates a positive result in all ELISA"s and all blots. A score between 0-40 indicates that not all possible combinations yielded a positive result (i.e. disagreement between various ELISA x blot combinations). Of this small sample cohort, 20/31 (65%) either had a score of 0 or 40, indicating perfect agreement irrespective of the ELISA x blot combination used. Discordant interpretations were generated in the other 35%.

Figure 1

 1 Figure 1 depicts an examplevalues for the VIDAS and Immunetics C6 Lyme ELISA

  divergent sensitivity and specificity, and immunoblots for detecting anti-Borrelia antibodies have only limited agreement. Therefore, the choice of ELISA-immunoblot combination severely influences the number of positive results, making exchange of test-results between laboratories with different methodologies hazardous. The widespread availability of more specific and sensitive assays for the detection of anti-Borrelia antibodies will open the way for a reappraisal of the two-tier testing system. of Hygiene/College of American Pathologists Proficiency Testing Program. Journal of clinical microbiology 35 (3):537-543 8. Wormser GP, Carbonaro C, Miller S, Nowakowski J, Nadelman RB, Sivak S, Aguero-Rosenfeld ME (2000) A limitation of 2-stage serological testing for Lyme disease: enzyme immunoassay and immunoblot assay are not independent tests. Clin Infect Dis 30 (3):545-548 9. Jansson C, Carlsson SA, Granlund H, Wahlberg P, Nyman D (2005) Analysis of Borrelia burgdorferi IgG antibodies with a combination of IgG ELISA and VlsE C6 peptide ELISA. Clin Microbiol Infect 11 (2):147-150 10. Smismans A, Goossens VJ, Nulens E, Bruggeman CA (2006) Comparison of five different immunoassays for the detection of Borrelia burgdorferi IgM and IgG antibodies. Clin Microbiol Infect 12 (7):648-655 11. Wormser GP, Nowakowski J, Nadelman RB, Visintainer P, Levin A, Aguero-Rosenfeld ME (2008) Impact of clinical variables on Borrelia burgdorferi-specific antibody seropositivity in acute-phase sera from patients in North America with culture-confirmed early Lyme disease. Clin Vaccine Immunol 15 (10):1519-1522 12. Aguero-Rosenfeld ME, Nowakowski J, Bittker S, Cooper D, Nadelman RB, Wormser GP (1996) Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. Journal of clinical microbiology 34 (1):1-9

Table 1 - Performance of eight ELISA's in three patient groups ELISA manufacturer Antigen used for ELISA Number of positive samples (%) Total number of tested samples Figure 1 -ELISA test value in relation to immunoblot results for detection of anti-Borrelia antibodies

 1

Acknowledgements

This work has been presented at the 20 th ECCMID, Vienna, Austria April 2010. The authors would like to acknowledge Stephen Johnston for editing the final manuscript.

Patients suspected for Borrelia infection

Crossreactivity controls

Healthy controls

Diacheck/Moran