M Conte
email: mario.conte@enea.it

Supercapacitors technical requirements for new applications

Keywords: supercapacitors, designs, classifications, performances, new applications, technical requirements supercapacitors, designs, classifications, performances, new applications, technical requirements. * SMES = Superconducting Magnet Energy Storage

may come

Introduction

The need to improve efficiency and sustainability of energy systems in all the aspects from generation up to the end uses is motivating the research and development of new storage systems and methods. The variety of existing or projected applications ranging from consumer electronics, electric and hybrid vehicles up to large power generation plants and electricity distribution grids with improved power quality and larger introduction of renewable energy sources requires storage systems with extremely varying performance characteristics.

No ideal energy storage device and method are insofar available to meet all technical and economical requirements from a growing spectrum of applications. Electrochemical batteries, SMES (superconducting magnetic energy storage), pumped hydro, flywheels, CAES (compressed air energy storage) and hydrogen storage are alternative and/or competitive solutions for storing energy usually proposed.

The supercapacitors (SCs), also called ultracapacitors or electrochemical capacitors, are devices with a very high specific power and high capacitance, available for a long period of time with negligible deterioration, that have been initially proposed in small applications (memory back-up, cold start assist of thermal engines, storage systems for micro solar power generators) and now are proposed for high power/energy applications, such as hybrid and electric vehicles, power quality systems and smart grids. Since their initial discovery in the middle 1960's, SCs have received increasing interest and stimulus to progress by the growing demand of new specialized energy storage systems with high specific characteristics and long cycle life [START_REF] George | Final report to ENEA[END_REF][START_REF] Halper | [END_REF][START_REF] Burke | [END_REF].

The advancements in new materials and the rapid growth of more demanding storage systems in a variety of applications have created a lack of universally accepted definitions of these devices and, consequently, a real difficulty in describing developments and progress in the SC field. In the last few years, many novel configurations of SCs have been investigated often mixing up battery and SC components with different behaviors and performance characteristics, all aimed at modifying specific technical parameters to better meet the technical, and even economical, requirements for the various applications.

The different types of SCs and the large extent of new applications have been demanding for suitable definitions and performance measurements that require the development of specific standards and procedures. Basic parameters are well known and are mostly based on a simplification approach that represents the SC with an equivalent electrical circuit model with defined electrical components. Unfortunately, the progress in configurations has somehow modified the real behavior of the various SCs, by asking for an experimental effort to harmonize and make comparable the key parameters of the different SC types.

The various applications of SCs, alone or in combinations with batteries or other energy source, are also addressing design issues: micro-systems as those of watches or memory back-ups look at very low energy content but for a very long time, while large energy/power storage systems, such as those in transport means (hybrid vehicles with thermal engines or fuel cell generators) are more interested in SC devices with very high power capability in charge and discharge, lasting thousands of cycles.

This paper contains a brief survey of the history of the SC development, which is strongly related to the evolution of the types and related definitions. In the second part, an attempt is made to classify the various categories of present SC and to give some definitions to divide the recent scientific and technological advancements in identifiable groups. In the third part, the basic performance parameters are defined with a rough analysis of the existing testing procedures and standards to measure them. The fourth part gives some examples of the major and most interesting applications with description of technical requirements for some of them: the major emphasis is given to vehicle applications, which are receiving specific attention in recent years. Finally, in the fifth part, the market prospects that are positively seen by various companies are reported for the coming years.

Historical hints

The start of the SC technology is surely based on the work of Helmholtz, who first in 1853 recognized that the electrical charge could be stored not only on the surface of a conductor but also at "double-layer" interface between an electrode and an electrolyte [START_REF] George | Final report to ENEA[END_REF][START_REF] Kurzweil | Encyclopedia Electrochemical Power Sources[END_REF]. Subsequently, almost one century later at GE (General Electric), in a not completely clear way, and at Sohio (The Standard Oil Company, then BP America, a subsidiary of the British Petroleum), first potential products were studied and patented. These initial claims were mainly the byproducts of research work on carbon electrodes for fuel cells and novel batteries. The chief result at that time was the discovery of extraordinary high specific capacitances in high-surface-area carbons in molten salts or, preferably, in aqueous electrolytes. In practice, the "double-layer" capacitance at the most electrode-electrolyte interfaces, in the range of 5-50 microfarads/cm 2 , was greatly improved by the high specific surface areas (up to about 1,500 m 2 /g) of activated carbons. As a consequence, it was realized that capacitances of about some farads could be obtained in cells of 10-20 mL in volume: about 6 orders of magnitude with respect to the conventional capacitors. In the 1970's, there was a decade of enthusiasm and illusion in which different companies (Sohio, Corning Glass, NEC) tried to turn the electrochemical results of Sohio into a commercial product. Finally, in 1979, NEC introduced on the market the first carbon-electrode SC with the brand name "Supercap". Since then, with a growing interesting market, mostly covered by Japanese companies, the SC technologies continued to progress.

Alternative classes of SCs were, in parallel, researched and developed by trying to improve basic technical performances of commercial carbon-based SCs: the energy content, related to the low decomposition voltage of the electrolyte and/or to the low capacitance of the pure electrostatic charge transfer. At the beginning of 1980's, the investigation of the so called "pseudocapacitors" started: a novel device with the same technical features of an SC (as referred to specific capacitance), but working in a different way [1, 4 and 5]. The electrical charge was partially stored in the "double layer" and partially was the result of faradaic reactions (charge transfer of electron and protons) between electrode and electrolyte: the working mechanisms are moving from a conventional capacitor to a conventional electrochemical battery. These devices contain ceramic electrodes composed of transitional-metal oxides (e.g., iridium dioxide, ruthenium oxide and nickel oxide and a combination of these), deposited as thin coating on metallic substrates. Another class of SC, investigated and even marketed in 1970's, uses solid-state electrolytes, such as sodium-silver-iodide. These SCs were commercialized for a short time by Gould Ionics in a configuration having silver anodes and activated carbon cathodes with most charge storage at cathode-electrolyte interface: the low dissolution voltage (about 0.6 V) and the high cost of the cathode solid electrolyte material negatively impacted customer interest on this device, which was quickly withdrawn from the market.

Finally, in the last two decades with the introduction of high-voltage decomposition electrolytes (e.g., organic, ionic liquids) [6, 7 and 8], the improvement of activated carbon properties and the introduction of new configurations (increased combination of SC and batteries components) and the growth of market demand on specific high power devices, the classes and designs of SCs have significantly augmented with the creation of a sort of chaos [START_REF] Malak | Proc. 3rd European Symposium on Supercapacitors and Applications ESSCAP'08[END_REF] in the SC field with the proliferation of categories and definitions used for the description of these devices. Figure 1 briefly summarizes the chief milestones and the key dates for the progress of the SC technology in most chemistries and designs.

General classification

The SCs are basically working according to the same principles of conventional capacitors, both electrostatic and electrolytic, with much different technical properties: an SC has specific capacitance and energy 10 times greater than that of an electrolytic capacitor and 100 times than that of an electrostatic capacitor. SCs are the scientific and technological evolution of conventional capacitors towards electrochemical batteries with clear fundamental differences: the main one is that in the batteries the energy is stored by means of chemical reactions, while in SCs the energy can be stored by simply electrostatic charge transfer. In practice, the differences between The rapid evolution and developments of these devices have been creating serious difficulties in identifying and agreeing upon an internationally accepted nomenclature. Different definitions and terms have been used over the years to classify SCs. Initially, it was accepted to indicate these devices as electrochemical capacitors, to better distinguish them from the conventional capacitors, but this definition was hardly accepted by some companies for commercial reasons: the term electrochemical could be disqualifying for more conventional capacitors. Other terms for SC have been ultracapacitors, pseudocapacitors (or "Batcaps"), and even brand names from leading producing companies have been used. In the last two decades, the classification of the SC has been more related to the specific designs, the use of specific materials in the electrolyte (organic, aqueous, ionic liquids, solid) and/or in the electrodes (activated carbons, electronically conducting polymers, ceramic oxides, battery-like components).

An exemplary case of the ambiguity existing in the SC terminology is represented by the interpretation of the acronym EDLC, which was initially used to describe carbon-based SC: they are purely capacitive devices in which the charge and discharge processes are very rapid, due to the simple electric charge orientation of the double layer at the electrode-electrolyte interface. In reality, EDLC acronym is equally used for two different definitions: 1) Electric Double Layer Capacitor; or 2) Electrochemical Double Layer Capacitor. The former surely represents the more correct operation way for these types of SC, while the latter generates confusion in the attempt to introduce in the same device the concept of "pseudocapacitance", which is typical of SC devices with faradic processes, or to emphasize the fact that the charge reorientation happens at the electrochemical double layer [START_REF] Halper | [END_REF][START_REF] Kurzweil | Encyclopedia Electrochemical Power Sources[END_REF]8]. This double meaning is currently used in the scientific and technological literature [8,[START_REF] Malak | Proc. 3rd European Symposium on Supercapacitors and Applications ESSCAP'08[END_REF] and even in international standards [10,11].

For sake of simplification in the attempt to more clearly classify the different types of SCs, an adaptation of existing taxonomy is here proposed. All the existing commercial and under development SCs can be divided in three main categories: 1. Symmetric SC 2. Asymmetric SC 3. Hybrid SC With respect to the existing approaches, the three terms are more related to the cell designs, electrode materials and the fundamental operating principles (physical or chemical processes, or a combination of the two). With significant adaptation from [START_REF] Halper | [END_REF][START_REF] Kurzweil | Encyclopedia Electrochemical Power Sources[END_REF][START_REF] Miller | Encyclopedia Electrochemical Power Sources[END_REF], Figure 2 presents the three possible categories of the current and under development SCs.

Symmetric SC

Symmetric SCs are basically the historical EDLC (electric double-layer). The term "symmetric" stands for the use of electrodes made with the same material (for example, activated carbon as in the EDLC) with the same design and, practically, the same mass loading on both electrodes. The prevailing charge storage principle effectively working (either physical or chemical or a combination of the two) must be the same on both electrodes.

Two major SC types (or classes) can be included in the symmetric category: 1) EDLC; and 2) pseudocapacitors.

Electric double-layer capacitors

The electrical charge is only physically stored in the double-layer, naturally formed at electrode-electrolyte interface under voltage. These devices are highly reversible and can guarantee the longer cycle life, because no chemical reactions, and then material transformations, happen during charge and discharge cycles (pure electrostatic charge storage). The large increase in basic properties (capacitance and internal resistance) with respect to conventional capacitors is mostly due to the use of the double-layer charge storage at both electrodes, normally using high-surface-area materials such as activated carbons, with a much higher surface area (in the order of hundreds of m 2 /g) and much smaller thickness of double layers (in the range of 10 -10 m), compared to the electrode distance in conventional capacitors. Alternative materials, or carbon formulations, can be used for this type of SC, all based on double-layer non-faraidic capacitance, with different electrolytes (aqueous, organic, ionic liquids) and different electrode (activated carbons, carbon aerogels, carbon nanotubes).

Pseudocapacitors

A second class of symmetric SC is represented by supercapacitors. Such devices are composed by electrodes using active materials that work differently from EDLC to store electrical charge and, them, energy. In fact, only part of the charge storage is assured by the double layer between electrode and electrolyte, while a greater amount of charge transfer and storage is due to faraidic mechanisms (electrosorption, redox reactions, and intercalation). The latter process is able to more than double the energy density than that of EDLC. From the original ceramic materials (iridium dioxide, ruthenium dioxide, nickel oxide and their combinations), new materials with pseudocapacitive characteristics have been investigated in recent years, such as electronically conducting polymers and other metal oxides (vanadium oxide, manganese dioxide) [START_REF] George | Final report to ENEA[END_REF][START_REF] Arbizzani | [END_REF]14,15]. These devices have had in the past significant drawback in the use of previous metals, which made expensive their application. Conducting polymers (for example, by negatively and positively doping electrodes) may improve their economic attractiveness, while applications requiring limited cycle number may take advantage of the significant higher energy density of any type of pseudocapacitor.

Asymmetric SC

Asymmetric SCs are included in the most controversial category. Different views are described in literature about the term of asymmetric configurations, as a result of the significant developments in SC technology over the last 20 years. A first definition issued in 2002 clearly showed the uncertainty on how to classify new SC devices: the "asymmetric" supercapacitor is the supercapacitor, on polar electrodes of which processes different in their nature are carried out [START_REF] Conway | Proc. of "The 12th International Seminar on DLC and Similar Energy Storage Devices[END_REF]. As a representative example of this category of SCs, a redox reaction on one of the electrodes and charge-discharge of electrostatic charge storage in the electric double layer on another was then mentioned. In the same period, the term "hybrid" has been alternatively used to name such SCs. In order to improve clarity in the increasing variety of SC designs and chemistries, a clear distinction of the two terms "asymmetric" and "hybrid" might be advisable. This classification effort may be functional in practical selection and applications of these devices, but also useful in specifying technical characteristics, measuring procedures definition of standards and SC modeling. In this simplification approach, there is also the intention to take into account the recent advancements in electrolyte materials (such as non-aqueous, ionic liquids), whose applications can be further improved by optimizing the different maximum potential excursions achievable, separately, at the negative and positive electrodes [START_REF] Mastragostino | Encyclopedia Electrochemical Power Sources[END_REF]. In this category, it is thus possible to include all the modified symmetric SCs, in which there are changes in the electrolyte compositions and in the mass loading and geometry of the electrodes, without changing the active materials and the operating processes (physical or chemical).

Hybrid SC

Hybrid SCs are the most innovative solution to combine the better performances of different SC configurations. The basic idea is to substantially improve technical characteristics, and even reduce costs, of more conventional SCs by using the best geometry and materials for each component. In practice, the recent research and development activities have shown multiple combinations of positive and negative electrodes coming from various technologies, even from conventional electrolytic capacitors: the major technological challenge is to reduce the performance gap between SC and electrochemical batteries by using an electrode from one technology and the other electrode from the other technology. The basic design of a hybrid SC uses two electrodes made of different materials and, eventually, using different operating processes (faraidic and nonfaraidic). The investigated combinations are aimed at maintaining the long cycle life of EDLC and power capability of conventional capacitors, reduce the cost constraints of pseudocapacitors and try to increase specific performances (e.g., energy density) to get closer to those of batteries. Three main types can be considered in this category: 1) Hybrid electrolytic capacitors; 2) Composite; 3) Battery-like.

Hybrid electrolytic capacitors

A pseudocapacitive electrode, as those based on metal oxides, can be combined with the electrode of a conventional electrolytic capacitor. In this way, the high power characteristic of electrolytic capacitors is still saved, but the addition of the pseudocapacitance on one electrode substantially augments the specific capacitance of the device.

Composite

A second class of hybrid SC is represented by devices, combining the electrode of an EDLC with a composite electrode, using carbon-based material (for example, carbon nanotubes) that incorporates either a conducting polymers or a metal oxide [6,[START_REF] Du Pasquier | [END_REF]19]. Such devices are also called asymmetric hybrid SC.

Battery-type

These SCs put in the same device two completely different electrodes, coming from two different storage technologies: an SC electrode is then coupled with a battery electrode. Recent research has investigated different combinations of SC electrodes with electrodes derived from batteries: lead acid (lead oxides), alkaline (nickel oxides), lithium (titanate, manganese oxide and others) [20,21,22,[START_REF] Marumo | Proc. 3rd European Symposium on Supercapacitors and Applications ESSCAP'08[END_REF]. These devices are considered extremely interesting even if more research is required to reduce the main disadvantages related to the need to overcome battery-related limitations.

Basic performance definitions and measurements

The use of SCs is growing with more demanding technical performances to better meet specific requirements from applications varying from very small sizes, with limited energy content, up to the level of hundreds of kW in various vehicles (hybrid passenger cars, hybrid buses and trains) and for electric utility services. The variety of applications has also determined the need for clearly measuring the SC properties, which may differ when they are used in different operating conditions. It is well know that technical properties of SCs are from being ideal and may be greatly affected by frequency, temperature, current and, in same case, by the category (symmetric, asymmetric and hybrid may have different reactions to the various working conditions).

International standards have been already defined [10,11] for measuring and determining the key technical properties of SCs, but these standards are referred to small-size applications in electronic equipment, such as, back up memories for consumer products. In addition, the current standards consider only EDLCs.

In parallel, testing procedures have been also developed over the years to respond to more specific demands of SC testing for automotive applications [START_REF] Miller | Electric Vehicle Capacitor Test Procedures Manual[END_REF][START_REF]FreedomCAR Ultracapacitor Test Manual[END_REF][START_REF]Specification of Test Procedures for Supercapacitors in Electric Vehicle Application[END_REF] and, more recently, related standards are under preparation.

An ideal SC can be represented with a first-order electric equivalent circuit, as described in Figure 1. It is comprised of four ideal circuit elements, which include a capacitor C, a series resistor R s , a parallel resistor R p , and a series inductor L. R s represents the equivalent series resistance (ESR) and accounts for the power loss during charge and discharge. R p is the parallel resistance responsible for the energy loss due to capacitor selfdischarge. This model is the simplest one and also the most adequate for low and medium power applications. In high power applications the non-ideal behavior of electrochemical capacitors cannot be neglected, depending on temperature, current, power, frequency, in case of alternate current systems, and also on SC category. The charge-discharge curve for the SC, represented by the circuit in Figure 3, is presented in Figure 4.

where, V R is the rated voltage (V) V 1 is the start voltage (V) for calculation V 2 is the end voltage (V) for calculation ∆U 3 is the voltage drop (V) T CV is the constant voltage charging duration (s) t 1 is the time elapsed from start to reach voltage V 1 t 2 is the time elapsed from start to reach voltage V 2 Figure 4 assumes that the charge and discharge behavior of an ideal SC is practically linear. In reality, the charge and discharge curves are nonlinear and are normally dependent on the current and the rated voltage. Consequently, most SC performance parameters must be measured according to the working conditions. Looking at the applications, it is also apparent that only some performances must be necessarily known and measured to be able to design the SC system and incorporate it in the final application.

The basic SC performance data to be necessarily known in any application are: capacitance, ESR and cycle life. Other technical performances are more useful in certain applications, particularly when large amount of energy and power is involved: self-discharge, mass and volumetric energy and power density and efficiency.

It is important to outline that for medium to high power applications, the basic performances are measured and calculated during charge phase, and not only during discharge, because the SC behavior changes and may affect the design and functioning of the system.

Capacitance

The capacitance of an SC is a intrinsic constant property, measured in Farad (F), and is related to the amount of electrical charge Q (in coulomb) effectively stored in the SC, when it is at the rated voltage V R , according to the following equation:

(1) The standardized method proposed to measure the capacitance is called "constant current discharge method" and is based on the discharge curve in Figure 3. With this method, the capacitance is calculated with the following equation: [START_REF] Halper | [END_REF] where I (in A) is the discharge current. This method has been mostly based on the assumption that the discharge behavior of the SC remains linear. The values of V 1 and V 2 can differ from various procedures and standards and can be stated as a fixed quotient of the rated voltage. In current standards for electronic equipment, V 1 = 0.8 V R and V 2 = 0.4 V R , while in testing procedures for electric vehicles, V 1 = 0.6 V R and V 2 = 0.4 V R .

An alternative method can be applied in order to minimize the potential voltage differences due to non linearity of the discharge curve. The "energy conversion capacitance method" is then suggested to calculate the SC capacitance and is based on the energy equation: where E, in Joule (J), represents the amount of energy discharged effectively when the SC voltage changes from V 1 to V 2 , also in this case these voltages can be fixed values of the rated voltage.

ESR

The internal series resistance, also called Equivalent Series Resistance (ESR), is the value of the element R s in the first-order circuit, as described in Figure 3. Different methods have been applied until now and new ones have been proposed for measuring the ESR. It corresponds to ohmic resistance associated with the voltage drop in the discharge curve represented in Figure 4. The ESR value does not depend on the voltage at which the SC is charged of discharged. The most common measuring method is called "current interrupt": the variation of voltage due to the sudden change in current is measured. The ESR is calculated with the following formula: [START_REF] Kurzweil | Encyclopedia Electrochemical Power Sources[END_REF] where ESR, in ohm, represents the ohmic resistance of the SC when the SC voltage changes from V i to V f , with a quick variation of current I.

Cycle life

It is generally believed that SCs do not show appreciable deterioration over a long period of time and under hundreds of thousands of charge and discharge cycles, even if it is fully discharged at a voltage near to zero. In reality this is true for the EDLC, but it is not the case for SC, in which faraidic processes are implied to store charge. The ageing of SCs is related to a variety of processes, which depend on the SC design and the operating conditions, then, from the applications. A critical aspect in the cycle life determination is the definition of the end of life criteria: some characteristics, such as capacitance, ESR and rated voltage, of the SC changes significantly and can be used to state the end of the life. Cycle life test procedures are normally based on a constant current profile. The SC are charged and discharged at constant current between the rated voltage and a defined end-of-discharge voltage arbitrary selected, as a percentage of the rated voltage (normally, one half or one-third of the rated voltage). Periodically, after a defined number of complete charge and discharge cycles, the basic characteristics of the SC are measured and the variations are estimated. The measurement is terminated whenever the capacitance (or the energy) decreases for at least 20 or 30 % with respect of the initial one [START_REF] Miller | Electric Vehicle Capacitor Test Procedures Manual[END_REF][START_REF]FreedomCAR Ultracapacitor Test Manual[END_REF][START_REF]Specification of Test Procedures for Supercapacitors in Electric Vehicle Application[END_REF].

Peak power

The high power capacity is one of the peculiar features of the SCs and must be clearly defined and accurately measured or calculated. It is out of the scope of this paper to examine all the possible definitions of peak power, which are normally used. There are anyhow some definitions, which are normally presented in product data sheets or even in analyzing application requirements. One common approach to calculate the peak power of an SC is based on the concept of maximum power transfer: considering the simplified circuit of Figure 3, it represents the condition where a load with a resistance equal to the ESR (R s ), then called matched load, is attached to the SC. Then, the initial peak power P peak delivered to the load is calculated from the following equation:

(

Unfortunately, this maximum power is transferred to the matched load with only 50% of power transfer efficiency, and, then one-half of the stored energy in the SC is dissipated as heat within the SC. Alternative methods for measuring peak power consider the maximum power achievable in 5 sec to discharge an SC from V R up to V R /3 [START_REF]Specification of Test Procedures for Supercapacitors in Electric Vehicle Application[END_REF], or the maximum power associated with the maximum current at a given voltage, according to the equation: (6) where I MAX is selected in a way to guarantee an energy efficiency of 95%, both in charge and discharge. For such case, the peak power is given by: F o r P e e r R e v i e w (7) where EF is the energy efficiency and V R is the rated voltage.

Specific energy and efficiency

The ideal value of the electrical energy stored in an SC is calculated by: (

) 8
where C is the capacitance and V R is the rated voltage. In practical uses, the complete discharge up to a voltage equal to zero is impossible and, consequently, a useable energy content is defined, which considers the energy delivered when the SC is discharged in a defined manner (e.g. at constant current or at constant power) from the rated voltage V R up to V R . For most application, the maximum discharged energy is given by: (

The energy efficiency is defined according to the definition of delivered energy as the ratio in percent of the energy delivered by an SC to the energy that was supplied to it during a defined cycle.

The specific values of energy and power can be easily obtained by dividing the measured or calculated values of energy and power with the overall weight of the SC.

SC new applications areas

At beginning of its commercial history, about three decades ago, SCs were mainly produced in limited sizes for small, in capacitance and voltage, applications. The preferred uses over the years still remain the prevailing applications in consumer electronics with SC devices containing a few unit cells with a total voltage below 11 V and an overall capacitance up to a Farad. The main function in low voltage, power and capacitance applications is to protect memories and simply CMOS circuits in video and audio systems, in electronic toys and watches, in security and alarm systems, in PC motherboards, in home telephones, and in smoke detectors. The technical requirements for such applications are from 50 mF up to 1 F with an overall voltage of about 5,5 V [8].

The growing demand of high performance power devices with enabling the development is open up the SC application to new industrial sectors with brand new market areas, with much larger SC systems: industrial and residential applications, transportation and conventional and renewable electricity grids. The demanding technical and economical requirements for these new application areas have fostered the variety of scientific and technological breakthroughs in the SC technology with a large spectrum of technical characteristics [START_REF] Burke | Encyclopedia Electrochemical Power Sources[END_REF]. Table 1 gives an update view of the state-of-the-art of current SC commercially available and under development, practically covering all the categories [START_REF] Burke | Proc. 24th International Electric Vehicle Symposium EVS-24[END_REF]. These applications normally require large or very large SC devices with technical requirements which are application-specific and, often, may greatly vary in size and performances in the same application area.

Currently, the use of large SCs is really promising with many prototypes and demonstration projects, seconded by a lot of simulation analyses covering applications from tens of watts up to tens of kW. The major concern is now of economical nature with the high cost of SC devices that cannot be clearly recovered with the interesting savings and advantages associated with their applications.

Anyhow, new applications areas for large SCs entail the following sectors:

• Industrial applications;

• Electric utility applications

• Automotive applications

In each of these sectors, different uses can be envisaged with promising potentialities for SCs. Most of these applications require high power storage systems which may be eventually coupled with a high energy storage system. The basic idea is to use the most appropriate technology for each function to be performed, considering both technical and economical aspects. Each applications analysis needs to clearly identify technical requirements and costs for design evaluation.

Industrial applications

In the industrial, residential and business building sectors, there are increasing demands for storage systems for back-up needs and for improving energy efficiency of systems aimed at carrying and elevating masses. The most interesting applications are the uninterruptible power supply systems (UPS) and the integration of storage systems in civil and industrial movers, such as elevators and cranes.

UPS

The use of electronic devices, the large diffusion of computers rooms and even the extended use of software controlled industrials processes are sensitive loads, all requiring back-up devices in case of emergency and fault conditions. The UPS with battery energy storage are devices very well developed and applied to sensitive loads and in critical events. The need of power quality improvement and number loads requires uninterruptible supply and is rapidly growing, due to the significant economical losses, due to lack of continuity and quality in power supply. The battery is the source of energy which is delivered to load usually, but the power capability of electrochemical batteries is not comparable with that of SCs. Depending on the specific operating conditions and failure risks the UPS are addressing, two different designs are now proposed [START_REF] Chlodnicki | [END_REF]: 1) Combined SC and battery UPS. By combining an SC with a battery-based UPS system, the life of the batteries can be extended. The SC gives power during short-duration interruptions and voltage sags. The batteries provide energy and power only during the longer interruptions. This reduces the cycling duty on the battery and extends its life. 2) Only SC UPS. For sensitive loads needing only a few seconds or tens of seconds of ride-through, an UC system with no batteries is feasible and should have very low maintenance costs.

The technical specifications of SCs in UPS are consistent with existing technologies, because in this application the SC device has to provide power for a few seconds. About the cost, this strongly depends on the associated benefits and the power quality requirements.

Elevators and cranes

In principle, elevators and cranes are devices that vertically move masses by consuming electrical energy. In general, conventional elevator and crane systems are not capable of recuperating energy during downward motion for further use, even if, theoretically, with exclusion of friction losses, most of the energy spent for elevating masses can be recovered during descending phase. These devices cause huge energy losses and very high acceleration currents. SC may store large amounts of energy in a very short period of time and to release high currents on demand: exactly the key functions required by these applications. Many studies and demonstrations have been performed outlining the advantages of using SC in such systems [30,[START_REF] Iannuzzi | annual Conference of the IEEE Industrial Electronics Society in IECON[END_REF][START_REF] Casadei | IEEE International Symposium on Industrial Electronics[END_REF]. SC devices with power between 20 and 100 kW (for a discharge time up to 50 s) have been designed and even used with interesting practical and economical implications. In the practical use in a gantry crane for ship unloading, the integration of an SC bank (100 kW for 42 s) with a diesel generator has resulted in an overall fuel reduction per unit time of 38%, a yearly consumption reduction of $ 24k, and an yearly reduction of polluting emissions of 40%.

Electric utility applications

Progress in SC systems has made them suitable for high-power applications with growing interest from electric utilities, which are looking to these devices for performance improvement and reliability in a variety of areas, with much higher power levels and with voltage up to 600 V. The key peculiar features of SC are extremely appealing for a variety of applications in electricity grids: fast response time in millisec, high energy efficiency (more than 95%), high power density and long calendar and cycle life. Various functions can be then performed by SC devices in electric grids, such as, for example [START_REF] Ayad | [END_REF][START_REF] Eyer | Energy Storage for the Electricity Grid: Benefits and Market Potential Assessment Guide[END_REF]: 1. Transmission line stability. The stability of a transmission system by adding energy storage. This serves to dampen oscillation through the successive generation and absorption of real (as opposed to reactive) power.

There is also transient stability -the stability required after a utility event (loss of substation or major line). During a transient event, achieving stability requires a substantial capability to absorb energy quickly. This is somewhat analogous to "dynamic braking" because generator turbines must be slowed. A typical specification is 100MW with 500 MJ (< 5 sec). 2. Spinning reserve. This is the generation capacity that a utility holds in reserve to prevent service interruptions if a generator fails. An ultracapacitor system can be built to supply power during the interruption, until quickstart diesels begin to supply power. A typical specification is 20MW to 100MW and 300 MJ to 1500 MJ. 3. Area and frequency control. The lack of matching between electrical energy production and energy consumption (including losses) appears as a frequency variation. An SC system, thanks to its fast response time, would be considerably more effective than a generating plant in supplying frequency regulation. A system based on SCs can absorb or supply energy as required, freeing other generation sources from frequency regulation or tie-line control duties. A typical specification is 100 MW to 1000 MW and 0.1 MWh to 10 MWh. Voltage regulation is the ability of the grid to maintain stable the voltage in any point of a transmission line within 5%. Voltage control devices are capable of regulating local-area voltage or regulating power flow without a significant amount of energy storage. The need for voltage regulation and power flow control is site-specific and is compatible with SC properties.

Smart grids

"A smart grid uses digital technology to improve reliability, security, and efficiency (both economic and energy) of the electric system from large generation, through the delivery systems to electricity consumers and a growing number of distributed-generation and storage resources" [START_REF]The Smart Grid: An Introduction[END_REF][START_REF] Belyakov | the 16th International Seminar on Double Layer Capacitors and Hybrid Energy Storage Devices[END_REF]. The integration of renewable energy source and the overall regulation of the electric system can be aided by using energy storage devices. There are some functions in the smart grids particularly suitable for the features of SC systems. Table 2 summarizes the key requirements and the most suitable storage systems, associated with the main functions.

Automotive applications

In the transport sector, the use of SC devices has been proposed for a variety of vehicles and transport systems from small ones (light-duty commercial and passenger cars) up to heavy-duty systems, such as, trucks, buses, trams up to trains. The proposed applications for these devices range from very low energy content for very high power services (engine starts) up to more energy-intensive service in hybrid vehicle configurations combining conventional thermal engines and, even, more advanced electrical generators, such as the fuel cells. Even in these applications, the SC systems can be act alone or combined with a battery. The advantages to use SC devices are many: the availability of more power; the drivetrain efficiency is substantially increased because of the higher share of regenerative braking energy; the smoothing action during charge and discharge can be beneficial for the battery because reduces stress and improve cycle life. The potential positive effects of SC systems have stimulated efforts in public research program and industries. Technical requirements, at least for road transport applications, have been, in some cases, already defined and represent the current frontier for SC development. Table 3 summarizes the technical and economical requirements, defined in the FreedomCar Program in the USA, for the use of SC in 3 different modes: mild-hybrid (only start-stop function) at 12V and 42V and power-assist operation in hybrid vehicles.

Starting engines

Diesel fueled engines have problems to start at cold temperature (up to -40°C). The problem is mostly related to the viscosity of lubricating oil at low temperature that requires high cranking torque with decrease in temperature. Lead acid batteries are presently used in vehicles for starting engines, but at low temperature the power capability of these batteries is very low and with difficulties in supplying high discharge current necessary to crank a cold engine [START_REF] Sojref | Proc. 3rd European Symposium on Supercapacitors and Applications ESSCAP'08[END_REF]. Furthermore, this poor performance at low temperature in lead acid battery is further reduced with age, strongly affecting the ability to sustain this current during extended engine start. To overcome this problem, an SC device has been proposed in conjunction with battery to supply cranking current at low temperatures.

Electric and hybrid vehicles

In case of electric and hybrid electric vehicles, SC devices can be usefully applied to carry out different functions: 1) peak power storage for acceleration and recovery of energy during braking/deceleration; 2) peak shaving of peak demands on batteries; 3) cost reduction of electricity generators, such as fuel cells, by optimal sharing of power size according to the duty cycles. The advantages of using SC are various: 1) the life of battery is less affected by transient operating conditions like acceleration and deceleration because battery cannot be charged or discharged at high rate, reducing battery replacement needs during vehicle life; 2) the energy efficiency of the vehicle may be improved because there is a better charge capacity of SC with respect to battery at high charge rates; 3) the cost per power unit of electrical generators, as fuel cells, is significantly higher than that of SC, allowing for a power downsizing and cost reduction in drivetrain production. As already indicated in Table 3, the technical and economical specifications for some of the possible applications of SC devices in vehicles have been already stated [START_REF] Burke | Encyclopedia Electrochemical Power Sources[END_REF]. In general, for such applications, the overall energy content and power for SC devices may vary from 50 Wh up to 2000 Wh, depending on the configuration with or without batteries and the size of the vehicle.

Fuel cell hybrid vehicles

In 2000 Honda showcased a prototype fuel-cell vehicle FCXV3 with high-pressure hydrogen tank and SC power bank. The lay-out of the FCX is in Figure 5. The SC bank supports acceleration and regenerative braking.

The definition of the application requirements for the developed SCs according to determined duty cycles and the possible role and impact of SCs in FCVs have been also investigated by means of mathematical models [38]. A reference van, named NEO (No Emission Outfit) has been used. It is a series-hybrid electric prototype, developed by Micro-vett (Italy), of the 'range extender' type, with a storage battery able to supply the traction power, while a FC system recharges the battery and extends the vehicle range, well beyond that available with the battery alone. The FC system power is lower than that required for moving the vehicle, with 30-kW continuous power and a 60-kW peak power (for 2 minutes). The rated powertrain voltage is 280 V, corresponding to the voltage of the 64Ah Zebra (Na-NiCl 2 ) battery system. The simulation work has been concentrated on simulating a modified vehicle configuration with the addition of the SC systems in order to reduce maximum current and peak power and extend cycle life of the battery system. Table 4 clearly presents the possible hydrogen savings under different duty cycles by using an SC integration in the NEO van.

Heavy duty and large transport systems

SC applications have been also proposed in very large transport system as hybrid transit buses, trams and trains.

Hybrid buses, trolley buses and trams

ISE Corporation produced a hybrid bus with a 360 V SC device to support a 225 kW electric motor, propelled by alternative generators (gasoline and diesel engines and even fuel cells). The small energy storage SC unit with about 0.5 kWh assists only acceleration of the vehicle. More than 100 ISE buses have been produced and are in normal daily service in USA.

SC units have been also proposed for trolley buses and trams in order to improve energy efficiency and minimize braking failure in both cases with or without feeding lines. Vossloh Kiepe in Germany has developed a trolleybus with an SC unit (Figure 6). The SC bank supports acceleration and regenerative braking. It operates without contact wires for 500 m. The SC unit is composed of 288 cells, has a capacitance of 2600 F and a total energy content of 648 Wh. The energy saving achieved with the SC unit is between 23-34 % with respect to the same vehicle without SC unit.

A similar concept of using trolleybuses with periodic service without feeding contacts has been proposed in Italy with the project "ZeroFilobus": SC units onboard of are charged during idle time at stop stations with fast charge schemes. A 24% of energy savings is estimated for this application.

Finally, an onboard SC storage unit is considered to be a promising tool for railway rolling stock. Electric trains can ran without feeding lines and diesel electric trains can absorb and use regenerative power. In case of electric trains with feeding lines, energy storage devices are useful to prevent regenerative energy failure at braking [39]. 6 Market prospects Today, commercial SCs are available from a number of countries, such as Australia, France, Japan, Russia, South Korea and Russia. The interesting market prospects envisaged for the new application areas are partially negatively counterbalanced by the high cost of the SC, which has delayed widespread diffusion. Nevertheless, the progress in the technologies and the manufacturer increased efforts are going to reduce cost and extend technical performances to a larger number of applications.

Table 5 presents the average trend of the SC costs, that clearly shows the specific cost per unit capacitance has been greatly reduced in 15 years (about 150 times) as well as the cost per energy unit (about 220 times smaller).

The SC market has been steeply growing in the last 20 years, even if it is still remained a few percents of the overall capacitor market. In 1989 about 75 million units were commercialized with total revenue of about $40 million. In 1999, about 201 million units were produced with a total value of about $ 115 million, while in 2004 there was a further increase until 660 million units with a related value of $ 276 million. The prevailing share (> 95%) of these markets has been for small SCs, but recent forecasts are quite optimistic with prospects of significant surge in the next almost 5 years. The expectations are to get close to about $877 million of worldwide sales by 2014 with a large share of sales represented by large SC applications, as those in transport and electric utilities, estimated to reach about $ 330 million.

Conclusions

The story and the recent developments of SCs have been thoroughly analyzed with the scope to understand and simplify classifications and sometimes misleading nomenclature. The significant progress in material science and in electrochemistry has made possible the realization of electrochemical capacitors with promising technical and economical properties suitable for a continuously increasing number of applications. SCs are now available in multiple designs that can be almost covering all the performance range existing between conventional capacitors and electrochemical batteries.

After the large applications in consumer electronics, in this article new interesting applications have been analyzed with the description of the targeted technical requirements and the relevant energetic and economical impacts. The described new applications areas refer mainly to large capacitance, power and energy SC systems and cover various energy-intensive sectors: industrial, transport and electricity grids. Various examples and demonstration project have been described to support this trend in SC applications. The outlook for larger diffusion of SC devices in these new application areas is also confirmed by market studies, forecasting an average yearly growth rate of about 27% up to 2014, with a largely increasing share (up to about 40%) of SC devices in large system applications.

These overall prospects and the associated advantages of using SC devices in new applications will be surely power near term and future efforts in research, development and, even, in production processes to further improve technical performances for a larger spectrum of applications and significantly reduce costs. Table 1 Technical performances of some present SCs [START_REF] Burke | Proc. 24th International Electric Vehicle Symposium EVS-24[END_REF].

F

Device or producer

Rated voltage V R (V) C (F) ESR (mOhm) Specific Energy (Wh/kg) (1)
T CV 電 圧 (V) V R V 1 V 2 ∆ V 3 Voltage (V) Time (s)

  are not so sharp, because the last SCs are trying to use properties and performance characteristics of both devices.

4 .

 4 Voltage regulation.

Fig. 1

 1 Fig. 1 Key milestones in the development of SC technologies. Fig. 2 Classification of SC categories and classes. ... Fig.3The first-order electric equivalent circuit of a capacitor. Fig.4The typical charge-discharge curve for an EDLC. Fig.5. Honda FCX prototype. Fig.6SC unit in the Vossloh trolleybus.

  Figure 2

Figure 8 .

 8 Figure 6.

  [38] M. Conte, M. Pasquali, Proc. 24th International Electric Vehicle Symposium EVS-24, Stavanger, 2009. [39] M. Yano, M. Kurihara, S. Kuramochi, Proc. 3rd European Symposium on Supercapacitors and Applications ESSCAP'08, Rome, 2008. [40] www.electronicsweekly.com/articles.

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Energy density at 400 W/kg constant power, V R -1/2 V R (2) Power based on P MAX =9/16*(1-EF)*V 2 /ESR, EF=efficiency of discharge * Except where noted, all the devices use acetonitrile as the electrolyte. ** all devices except those with ** are packaged in metal containers, these devices are in laminated pouches. Technical characteristics of some key functions in smart grids.

	Specific	Specific	Weight	Volume
	Power	Power	(kg)	(L)
	(W/kg)	(W/kg)
	(95%)	Matched
	(2)	Load

Table 3 .

 3 United States Advanced Battery Consortium -FreedomCar Program. SC Requirements at the end of life for 3 different services.

	Page 15 of 23	Fuel Cells
	System Attributes	12V Start-Stop (TSS)		42V Start-Stop (FSS)	42V Transient Power Assist (TPA)
	Discharge Pulse	4.2 kW	2s	6 kW	2s	13 kW	2s
	Regenerative Pulse	N/A		N/A		8 kW	2s
			7 V		21 V		21 V
	Cold Cranking Pulse @ -30°C	4.2 kW	Min.	8 kW	Min.	8 kW	Min.
	Available Energy (CP @1kW)	15 Wh		30 Wh		60 Wh
	Recharge Rate (kW)	0.4 kW		2.4 kW		2.6 kW
	Cycle Life / Equiv. Road Miles	750k / 150,000 miles	750k / 150,000 miles	750k / 150,000 miles
	Cycle Life and Efficiency Load
	Profile	UC10		UC10		UC10
	Calendar Life (Years)	15		15		15
	Energy Efficiency on UC10 Load
	Profile (%)	95		95%		95%
	Self Discharge (72hr from Max. V)	<4%		<4%		<4%
	Maximum Operating Voltage (Vdc)	17		48		48
	Minimum Operating Voltage (Vdc)	9		27		27
	Operating Temperature Range (°C)	-30 to +52		-30 to +52		-30 to +52
	Survival Temperature Range (°C)	-46 to +66		-46 to +66		-46 to +66
	Maximum System Weight (kg)	5		10		20
	Maximum System Volume (Liters)	4		8		16
	Selling Price ($/system @ 100k/yr)	40		80		130
		Wiley-VCH

Table 4 .

 4 Hydrogen savings with the introduction of an SC device in the fuel cell van NEO by Micro-vett.

		Page 16 of 23
	Duty Cycle	H 2 savings (%)
	Urban Ece 15	32.3
	Real Micro-vett	4.0
	Hyzem	13.6
	Hyzem_70	19.6

Table 5 .

 5 SC cost trend [40].

	Page 17 of 23
		Specific capacitance	Specific energy
	Year	cost,	cost,
		$/Farad	$/kJ
	1996	0,750	281,55
	1998	0,400	151,23
	2000	0,100	32,00
	2002	0,023	7,51
	2006	0,010	2,85
	2010	0,005	1,28

Wiley-VCH Fuel Cells


Holger ; Thiele

Marcel Du Moulin

Katarzyna Barczyk

Christel George

Wolfram Schwindt

Gudrun Nürnberg

Michael Frosch

Gerd Kurlemann

Johannes Roth

Peter Nürnberg

Marcel Du Moulin

Gerhard Kurlemann

Frank Rutsch
email: rutschf@ukmuenster.de

Dr Med Frank Pd

Rutsch

Aicardi-Goutières Syndrome Caused by the Arg164X Mutation in SAMHD1 Are Associated with Altered Cytokine Expression

Keywords: cytokine, cerebrovascular stenosis, stroke, samhd1, immune response, interleukin-8 cytokine, cerebrovascular stenosis, stroke, SAMHD1, interleukin-8, immune response

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

INTRODUCTION

Aicardi-Goutières syndrome (AGS) (OMIM # 225750) was first described in 1984 as an early onset familial encephalopathy inherited autosomal recessively (Aicardi and Goutières, 1984). It is a multisystemic disorder resembling intrauterine viral infection [START_REF] Goutières | Aicardi-Goutières snydrome[END_REF].

Patients present with psychomotor retardation, progressive microcephaly, dystonic posturing and spasticity as well as chilblain-like skin lesions [START_REF] Tolmie | The Aicardi-Goutières syndrome (familial, early onset encephalopathy with calcifications of the basal ganglia and chronic cerebrospinal fluid lymphocytosis)[END_REF]. Congenital glaucoma may be present (Crow et al., 2000). The syndrome manifests in infancy and death often occurs early in childhood in a state of decerebration. However, milder forms and survival into early adulthood have been reported [START_REF] Goutières | Aicardi-Goutières Syndrome: An Update and Results of Interferon-α Studies[END_REF][START_REF] Rice | Mutations involved in Aicardi-Goutières syndrome implicate SAMDH1 as regulator of innare immune response[END_REF].

Intracerebral calcifications, specifically at the site of the basal ganglia, cerebral atrophy, and leukodystrophy detected by cranial computed tomography (CT) scan are the most important characteristics [START_REF] Goutières | Aicardi-Goutières snydrome[END_REF]. Other diagnostic criteria include chronic lymphocytosis in cerebrospinal fluid (CSF) [START_REF] Goutières | Aicardi-Goutières snydrome[END_REF] and elevated levels of interferon-α (IFN-α) in CSF and serum [START_REF] Lebon | Intrathecal synthesis of interferon-alpha in infants with progressive familial encephalopathy[END_REF]. However, elevation of IFN-α levels diminishes with time [START_REF] Goutières | Aicardi-Goutières Syndrome: An Update and Results of Interferon-α Studies[END_REF] or may be absent [START_REF] Blau | Cerebrospinal fluid pterins and folates in Aicardi-Goutières syndrome -A new phenotype[END_REF][START_REF] Wassmer | Elevated pterins in cerebral spinal fluid -biochemical marker of Aicardi-Goutières syndrome[END_REF]). In such cases, elevated pterins in CSF can be a useful diagnostic marker [START_REF] Blau | Cerebrospinal fluid pterins and folates in Aicardi-Goutières syndrome -A new phenotype[END_REF][START_REF] Wassmer | Elevated pterins in cerebral spinal fluid -biochemical marker of Aicardi-Goutières syndrome[END_REF]). Furthermore, thrombocytopenia and elevated transaminases may be found [START_REF] Goutières | Aicardi-Goutières Syndrome: An Update and Results of Interferon-α Studies[END_REF].

Mutations in several genes have been found to be associated with AGS including TREX1, a gene on chromosome 3p31.21 encoding a DNA exonuclease (Crow et al., 2006a). Mutations in three genes encoding the ribonuclease H2 enzyme complex, RNASEH2A on 19p13.13, RNASEH2B on 13q14.3, and RNASEH2C on 11q13.2, have also been described in the pathogenesis of AGS (Crow et al., 2006b). Early onset of symptoms is associated with TREX1 mutations, while mutations in RNASEH2B lead to a late-onset presentation, a milder phenotype and reduced mortality [START_REF] Rice | Clinical and Molecular Phenotype of Aicardi-Goutières Syndrome[END_REF]. Most recently, mutations in SAMHD1 on 20q11 were identified in AGS [START_REF] Rice | Mutations involved in Aicardi-Goutières syndrome implicate SAMDH1 as regulator of innare immune response[END_REF] and phenotypic overlap with systemic lupus erythematosus has been described [START_REF] Ramantani | Expanding the phenotypic spectrum of lupus erythematosus in Aicardi-Goutières syndrome[END_REF].

We report on four affected siblings, two males and two females, born to consanguineous parents of Turkish origin (Table 1). The patients presented with an unknown multisystemic, neurodegenerative disorder. Their main clinical symptoms, including stenoses of intracranial vessels, stroke and glaucoma, could not be linked to any previously described syndrome. Therefore, using genomic DNA from the four affected siblings, we performed genome-wide homozygosity mapping and next generation sequencing and identified a mutation in SAMHD1 in all four patients. As this gene has been most recently described in AGS [START_REF] Rice | Mutations involved in Aicardi-Goutières syndrome implicate SAMDH1 as regulator of innare immune response[END_REF], we retrospectively confirmed the diagnosis by demonstrating intracerebral calcifications on CT scans in all four patients and elevated pterins in CSF in three of them.

CASE REPORTS

Patients were included in the study after obtaining written informed consent and the study was approved by the ethical committee of Münster University. Clinical findings of all patients are summarized in

Patient 1

The male patient was the second child born to consanguineous parents originating from Turkey. Six siblings (three male and three female) are asymptomatic. At one year of age he was diagnosed with meningitis.

He was of small stature and had reduced mental capabilities. His communication skills were sufficient, and cooperation was adequate. He was tetraspastic and mobile with support of a walking aide only. He developed glaucoma and cataract on his left eye, eventually leading to blindness. Furthermore, precocious puberty had been noted. His skin was unremarkable. Thyroid hormone levels were normal.

Magnetic resonance (MR) angiography, performed at age 37, showed irregular vascular structures of the left medial cerebral artery. At age 40, a CT scan showed symmetrical calcifications of the basal ganglia and of the white matter as well as global cerebral atrophy. CSF analysis revealed an increased cell count and elevated protein and lactate levels. Because of pathological MR findings in combination with the CSF study, the patient was thought to suffer from tuberculous meningitis and was treated with tuberculostatic drugs for 60 days. The clinical course was complicated by respiratory insufficiency, secondary to nosocomial pneumonia. The patient later developed acute respiratory distress syndrome and acute renal failure. He died of severe sepsis at 40 years of age.

Patient 2

The male patient was the fifth child of the family. He was born after an uneventful pregnancy (birth weight was 3,300 g, length 50 cm, head circumference 35 cm, Apgar 10/10/10). Congenital glaucoma was diagnosed.

At 3 months of age, motor development was delayed. There was no fixation at the age of 1 year and buphthalmos was noticed. At 2 ½ years of age, he could move using all four extremities and was able to sit without support. Walking was possible with an aide only. The patient suffered from a seizure with transient postconvulsive hemiparesis at 3 years of age. Another seizure occurred at 5 years of age. He lost speech at the age of 6 years and communicated using signs.

Symmetrical calcifications of the basal ganglia as well as cerebral atrophy were evident on cerebral CT at the age of 8 years. A maculous, erythematous, bluish-coloured skin lesion was first noticed on the left foot. Also, precocious puberty was noted.

At the age of 20 years, the patient was severely dystrophic with a height of 122 cm (< 3 rd centile) and a weight of 20 kg (< 3 rd centile). Additionally, multiple small cervical fibromas of the skin were present.

At age 22, mild lymphocytosis and increased protein levels were found in CSF. IFN-α was not analysed; CSF pterin levels were elevated.

Cataract of the left side was diagnosed at the of age 25 years.

A cranial magnetic resonance imaging (MRI) scan, performed at the age of 25 years, revealed marked stenoses of the supraaortic vessels. The internal carotid arteries on both sides and the arteries of the Circulus Willisii could only hardly be detected.

Because of a butterfly rash, chronic arthropathy and contractures, an undefined encephalopathy and positive serum antibodies against double-stranded DNA, systemic lupus erythematosus was suspected at this time and the patient was treated with immunosuppressants for several years. Under this regimen, he developed pneumonia and intracerebral hemorrhage ultimately leading to death at the age of 29 years. Shortly before the incident, he had recurrent ulcers and was treated with prostaglandin infusions. It can be suspected that this therapy contributed to the bleeding.

Patient 3

This female patient is the eighth child of the family. She was born at 38 weeks of gestation after an uneventful pregnancy (weight 3,600 g, length 51 cm, head circumference 35 cm, Apgar 9/10/10).

Symptoms were first noticed at age 4 when she presented with polyarthritis and inflammation of the eyes. With 9 years of age, she complained of increasing, non-tender restriction of movement of the fingers, elbows, hips, knees, and feet. Later, pain and contractures developed. She had reduced vision and glaucoma of both eyes. At that time, precocious puberty was noted.

At age 12, she developed erythema of both forearms after exposure to sun. Additionally, she was diagnosed with primary hypothyroidism.

At age 16, her feet were deformed, showed signs of arthrosis and were increasingly painful. She also suffered from gonarthrosis.

Similar to her siblings, she has a small stature. At age 17, her height was 136 cm (< 3 rd percentile) and her weight was 48 kg (3 rd percentile).

A CT scan, performed at age 25, showed bilateral calcifications of the basal ganglia (Figure 1A). At that time, CSF showed no pleocytosis. IFN-α was not elevated in CSF or serum. Pterin levels in CSF were increased (neopterin 93.3 nmol/l [9-20], biopterin 61.4 nmol/l [10-30]). MR angiography showed a fetal origin of the left posterior cerebral artery with a narrow segment without any pathological relevance. No inflammatory changes were seen on MRI of her right hand. Liver enzymes were mildly elevated (GOT 37 U/l [10-35], GPT 46 U/l [10-35], γ-GT 61 U/l [< 39]).

Mental capabilities are mildly impaired. The patient is able to communicate well and properly. She currently works as a waitress. The female patient is the ninth child of the family. She was delivered by cesarian section because of intrauterine tachyarrhythmia at 36 weeks of gestation (birth weight 3,200 g, length 53 cm, head circumference 34 cm, Apgar 6/9/9).

Since early infancy, she suffered from spasticity of the arms and legs. An ophthalmological investigation in the first days of life revealed no signs of congenital glaucoma. A cranial CT scan at the age of 2 years showed intracerebral calcifications and oligoclonal bands were found in CSF. She started crawling between age 3 and 4 years and was able to sit without support between age 4 and 5. She never developed expressive language, but she was able to understand language. Since age 8, she has been wheelchair dependent.

During the first six years of life, she had recurrent episodes of dermatitis of the flexor sides of the extremities. On the palm of her hands, she developed plaque-like lesions. Her right toe showed necrosis. Calcinosis cutis with axillary nodules was also noticed.

At age 8, she was diagnosed with precocious puberty.

Coxa valga developed on both sides at age 10 and at age 11 she suffered from increasing tenderness and stiffness of her joints, especially of the fingers. At age 11, she was also diagnosed with primary hypothyroidism.

At age 14, the patient was hospitalized with signs of cerebral ischemia, presenting with right-sided hemiparesis. A cranial CT scan showed signs of infarction of the left medial cerebral artery. In addition, calcifications of the basal ganglia and of the frontal white matter were noticed. MR angiography revealed a complex vascular pathology with complete occlusion of the left distal internal carotid artery and of the medial cerebral artery (Figure 1B). On the right side, there were severe stenoses of the internal carotid artery and of the proximal part of the medial cerebral artery.

Also at age 14, glaucoma of the left eye was diagnosed. Furthermore, a benign paravertebral perineuroma on the left side was detected.

A cerebral MRI scan, performed at age 17, showed marked cerebral atrophy. There were hyperintense lesions, interpreted as calcifications. Presumably resulting from the previous stroke, white matter lesions were evident in the left temporal lobe.

At age 20, her height was 147 cm (3 rd centile), weight was 65 kg (90 th centile) and head circumference 51 cm (3 rd centile). CSF analysis showed no pleocytosis, but increased protein and pterin levels (neopterin 161.1 nmol/l [9-20], biopterin 41.2nmol/l [10-30]) with mild dysfunction of the blood-brain barrier (CSF-to-serum albumin ratio 0.015 [< 0.006]). IFN-α levels were normal in CSF and serum.

MATERIALS AND METHODS

Homozygosity mapping

We used standard methods to isolate genomic DNA from peripheral blood samples according to the manufacturer's instructions (Puregene, Gentra Systems, Minneapolis, Minnesota, USA).

From each of the four patients, a DNA sample was processed according to the manufacturer's instructions (Affymetrix GeneChip Human Mapping 50K Assay Manual; Affymetrix, Santa Clara, California, USA). In brief, 250 ng of high quality genomic DNA was digested with HindIII and ligated to HindIII-adaptors. After PCR amplification, random fragmentation and labelling, samples were hybridised to the 50K array (GeneChip Human Mapping 50K Xba 240 array; Affymetrix) in a hybridisation oven (Affymetrix Hybridisation Oven 640; Affymetrix). Washes and staining of the arrays were performed using a fluidics station (Affymetrix Fluidics Station 450; Affymetrix), and images were obtained using a gene-chip scanner (Affymetrix GeneChip Scanner 3000; Affymetrix). Call rates had to exceed 98%. Allele calls were made using the BRLMM (Bayesian Robust Linear Model with Mahalanobis distance classifier) algorithm.

Parametric linkage analysis was performed using the program ALLEGRO [START_REF] Gudbjartsson | Allegro, a new computer program for multipoint linkage analysis[END_REF] assuming recessive inheritance with complete penetrance and no phenocopy. The disease allele frequency was set to 0.01%. Of all the 58960 SNP markers of the 50K array, we selected 12226 markers for the genome scan based on their minor allele frequencies and their distance to adjacent markers. The filtering was necessary to avoid markers in linkage disequilibrium to each other, which could give rise to false positive signals.

Haplotypes on chromosome 20 were reconstructed by the program ALLEGRO [START_REF] Gudbjartsson | Allegro, a new computer program for multipoint linkage analysis[END_REF] with microsatellite genotype data of all family members. Fragment analysis for microsatellite genotyping was performed on an ABI 3730 sequencer after PCR amplification with fluorescence labelled primers. Three of the markers had been established at the Cologne Center for Genomics and were registered in the UniSTS and Genebank databases as M20EK01= UniSTS:1233243/GF102162, M20EK02= UniSTS:1233244/ GF102163 and M20EK03= UniSTS:1233245/GF102164.

All data handling was performed using the user interface ALOHOMORA [START_REF] Rüschendorf | ALOHOMORA: a tool for linkage analysis using 10K SNP array data[END_REF]. Pedigree and haplotype drawing was performed with HaploPainter [START_REF] Thiele | HaploPainter: a tool for drawing pedigrees with complex haplotypes[END_REF].

DNA enrichment and next generation sequencing

To find the mutation, we used a strategy of DNA enrichment for the region of interest on chromosome 20 followed by complete sequencing using next generation sequencing technology. The sequence capturing was performed with two custom designed NimbleGen 385K Arrays from NimbleGen/Roche, each of them carrying 385,000 oligo-DNA features of 60 bp length. The array design was made to cover a region of 8.2 Mb on chromosome 20 at the positions 30.95 Mb -39.15 Mb -distributed over two arrays with the sequence data from UCSC human build hg18 and design algorithm 2.0 (http://www.nimblegen.com/products/lit/probe_design_ 2008_06_04.pdf).

In brief, the enrichment process started with fragmentation of 21 µg DNA per array by nebulisation into pieces of 250 bp to approximately 1 kb in size, followed by ligation of 22mer gSel3/gSel4 linkers for a post-enrichment library amplification step. The libraries were then hybridized to the NimleGen sequence capture arrays and subsequently unbound DNA fragments were washed away. After elution and recovery of bound fragments, the enriched pools were checked for quality and were amplified.

For sequencing on the Roche 454 Genome FLX sequencer, DNA libraries were constructed using the Roche shotgun library preparation method for low molecular weight DNA without prior nebulisation (GS FLX General Library Preparation Method Manual, Roche). In a next step, emulsion PCR amplification was performed as described in the Roche Manual (GS FLX Titanium emPCR Method Manual, Roche). The two libraries were then loaded onto a single 2-lane gasket PicoTiterPlate device (70 x 75 mm) and sequenced on a Roche GS FLX sequencer using standard Roche protocols.

Data analysis was performed using Roche's Newbler assembler V.2.0.0 -PostRelease-1 on the basis of human UCSC build hg18. An extended mapping of variants was performed on the ENSEMBL human build V.54_36p with a software developed at the Cologne Center for Genomics to rank variants according to their presumed functional relevance.

Sanger sequencing

The primer design for the amplification of exonic SAMHD1 regions was made by GeneExplorer (http://portal.ccg.uni-koeln.de/geneexplorer/). Cleaning of PCR fragments was performed with 3 U Exonuclease I and 1 U Shrimp Phosphatase in a total volume of 10 µl for 20 minutes at 37 °C followed by an inactivation step for 15 minutes at 85 °C. Cycle sequencing was performed with Applied Biosystems BigDye® Terminator v1.1 according to the vendor protocol (http://docs.appliedbiosystems.com/pebiodocs/04337036.pdf). Sequencing runs were performed on an ABI 3730 sequencer.

Nomenclature

Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen).

Cell culture

Human skin fibroblasts were isolated from patients and controls after informed consent. The cells were cultured at 37 °C in 5% CO 2 in MEM with Earl's Salt (PAA Laboratories, Pasching, Austria) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 200 IU/ml penicillin and 100 µg/ml streptomycin (all from Biochrom, Berlin, Germany). Confluent monolayers of human fibroblasts were stimulated for 4 and 24 hours with 50 µg/ml polyinosinic-polycytidylic acid (Poly(I:C)) or 10 ng/ml tumor necrosis factor alpha (TNF-α ) (both from Sigma, Munich, Germany). Control cultures were left untreated.

Real-time RT-PCR

RNA of control fibroblasts and of patient fibroblasts stimulated with Poly(I:C) was analysed in duplicate. cDNA was synthesized from 1 µg of total RNA using SuperScript TM II RNase-H reverse transcriptase (Invitrogen). Primers used for PCR analysis were as follows: GAPDH forward, 5'-TGCACCACCAACTGCTTAGC-3'; GAPDH reverse, 5'-GGCATGGACTGTGGTCATGAG-3'; SAMHD1 forward, 5'-AAAACCAGGTTTCACAAC-TTCTGC-3'; SAMHD1 reverse, 5'-TGCGGCATACAAACTCTTTCTGT-3'; IFNB1 forward, 5'-TCTGGCACAACAGGTAGTAGGC-3'; IFNB1 reverse, 5'-GAGAAGCACAACAGGAGAGCAA-3'; IL8 forward, 5'-CTTGTTCCACTGTGCCTTGGTT-3'; IL8 reverse, 5'-GCTTCCACATGTCCTCACAACAT-3'. Real-time RT-PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) as previously described [START_REF] Ehrchen | Glucocorticoids induce differentiation of a specifically activated, anti-inflammatory subtype of human monocytes[END_REF]. Results are expressed as a number of relative mRNA copies of each gene/10000 mRNA copies of GAPDH.

Western blotting

Fibroblasts were trypsinized, washed in PBS and lysed in High Salt Lysis Buffer (20 mM Hepes pH 7.9, 420 mM NaCl, 1.5 mM MgCl 2 and 0.2 mM EDTA) supplemented with EDTA-free complete protease inhibitors (Roche, Mannheim, Germany). The protein concentration of cell lysates was determined by the Bradford method. Equivalent amounts of protein (20 µg/lane) were subjected to electrophoresis on 10% sodium dodecyl-sulfate acrylamide gel and subsequently electroblotted to nitrocellulose membrane (Pierce, Rockford, IL, USA). The membranes were probed with rabbit antibody against SAMHD1 (Protein Tech Group, Chicago, IL, USA) followed by horseradish peroxidase-coupled secondary antibody using enhanced chemiluminescence. Membranes were directly reprobed with mouse monoclonal anti-β-actin antibody to verify equal loading and transfer of proteins (Sigma, Munich, Germany).

Immunofluorescence microscopy

Fibroblasts were cultured at a concentration 40 x 10 3 cells/ml on fibronectin (Becton Dickinson)-coated LabTec chamber slides (Nunc, Wiesbaden, Germany) to determine the cellular localization of SAMHD1 protein. Cells were stimulated with Poly(I:C) for 24 h or left untreated. Subsequently, cells were fixed with 10% paraformaldehyde in PBS for 10 minutes, permeabilized with pre-chilled acetone (-20 o C) for 5 minutes and washed in PBS. After blocking of unspecific binding sites with 1% BSA in PBS at room temperature for 1 h, staining with anti-SAMHD1 antibody (ProteinTech Group, Chicago, IL, USA) was performed. As secondary antibody FITC-conjugated goat anti-rabbit IgG (Dianova, Hamburg, Germany) was used. Cell nuclei were visualized by staining with 4,6-diamidino-2-phenylindole (DAPI) (Sigma, Munich, Germany). Coverslips were mounted on slides using Fluorescent Mounting Medium (Sigma, Munich, Germany). Immunofluorescence analysis was conducted using Carl Zeiss Axio Observer.Z1 microscope with AxioCam MRm camera and EC Plan-Neofluor Oil immersion (x40) lens using AxioVision Software.

RESULTS

Locus Mapping

To map the disease locus, a genome-wide linkage analysis was performed using the four affected family members. We found only one prominent linkage signal in the genome, namely on chromosome 20, showing a multipoint LOD score of 3.0 (data reviewed but not shown). The locus was confirmed by genotyping of microsatellite markers in all family members. Segregation of haplotypes of the critical interval on chromosome 20 is shown in Figure 2. Only the four patients were homozygous for the disease carrying haplotype in the linkage interval. The flanking markers M20EK02 and M20EK03 defined an interval of about 8.1 mega bases, where the mutation had to be looked for.

Sequence capturing and next generation sequencing

After masking of regions with low complexity, a total of 6.18 mega bases (Mb) of sequences could finally be used for the NimbleGen 385K array design. This represents a coverage of 75.3%. For the first library, 470,659 sequence reads comprising 135 Mb of sequences were recoverable. About 76% of the sequences could be mapped to the target region resulting in a total target coverage (≥1x) of 85%. The second library resulted in 650,943 reads and 199 Mb of sequences (65% on target with 86% target coverage). In total, 231 Mb of sequences covering the target region were available for mutation screening. With respect to coverage distribution, 29% and 24% of the mapped region were underrepresented with a factor of < 6 for the two enrichments.

Overall, 16,454 variants could be identified, most of them representing false positive signals due to low quality of sequence data. As a homozygous mutation was expected, a primary filter for variants with a frequency of at least 50% reduced the list to 6721. 755 variants remained after elimination of known SNP variants. Only 19 of these variants mapped to protein coding transcripts and led to an amino acid change as well. 5 variants were found to overlap with potential splice sites. By increasing the variant frequency limit to 90%, a total of 5 different amino acid changes in 4 different genes remained (reviewed but not shown) -one of them being the nonsense mutation c.490C>T (p.Arg164X) in exon 4 of the SAMHD1 gene (ENSP00000262878, GenBank NG_017059.1), which was seen in both enriched libraries in 12 of 13 reads.

Mutation screening

To validate the SAMHD1 c.490C>T (p.Arg164X) mutation, all family members were sequenced with the Sanger method. The mutation could be confirmed in all family members segregating the disease haplotype with the patients showing a homozygous mutation, while the parents and some of the unaffected siblings presented as heterozygous carriers (Figure 2). The c.490C>T (p.Arg164X) mutation could not be found in 96 Sanger sequenced Caucasian control samples.

SAMHD1 expression

SAMHD1 mRNA was present in all control fibroblasts as well as in fibroblasts isolated from patients with the p.Arg164X substitution, as assessed by real-time RT-PCR and dissociation curve analysis (data not shown). Analysis of protein expression in Western blot showed that SAMHD1 protein was expressed in all control fibroblasts, but was not detectable at protein level in lysates of fibroblasts isolated from affected individuals (Figure 3A).

Changes in SAMHD1 expression in response to Poly(I:C) and TNF-α

Western blots were performed to evaluate changes in expression of SAMHD1 protein in response to proinflammatory stimuli. Fibroblasts were stimulated with Poly(I:C) and TNF-α for 24 h. We found significantly increased expression of SAMHD1 in lysates of control fibroblasts treated with Poly(I:C) i.e., a 1.5 to 2.2-fold induction as revealed by densitometric quantification of SAMHD1 expression normalized to β-actin (data not shown), while the amount of SAMHD1 protein was not changed or only very slightly up-regulated after treatment with TNF-α. SAMHD1 protein was not detectable in lysates of patient fibroblasts (Fig. 3B).

Immunocytochemistry confirmed these observations. SAMHD1 protein was detectable in unstimulated control fibroblasts and its expression was increased in response to Poly(I:C) stimulation. Both in unstimulated and Poly(I:C)-stimulated cells SAMHD1 protein was mainly localized to the nucleus. In contrast, SAMHD1 protein was not detected in patient fibroblasts (data not shown).

The role of SAMHD1 protein in immune response against viral infection

We treated control fibroblasts and fibroblasts isolated from affected individuals with the TLR3-agonist and immunostimulant polyinosinic:polycytidylic acid (Poly(I:C)) to investigate how lack of SAMHD1 protein modifies immune response. Fibroblast response to Poly(I:C) was assessed by measuring the expression of proinflammatory factors using quantitative real-time RT-PCR. We did not detect any differences in basal and Poly(I:C)-induced expression of interleukin-6 (IL6), chemokine (C-X-C motif) ligand 9 (CXCL9) and intercellular adhesion molecule 1 (ICAM1) (data not shown). However, we found highly up-regulated basal expression of interleukin-8 (IL8) in patient fibroblasts as compared to controls (Figure 4A and4B). After 4 h of stimulation with Poly(I:C), similar amounts of IL8 transcript were induced in control fibroblasts and patient fibroblasts (Fig. 4C). After 24 h of Poly(I:C)-stimulation, IL8 was still up-regulated in control fibroblasts, but not in patient fibroblasts (Fig. 4D). High amounts of interferon-beta (IFNB1) transcript were found in control fibroblasts after 4 h of stimulation with Poly(I:C) (Fig. 4E) and up-regulation was still present after 24 h (Fig. 4F). IFNB1 was not induced in response to Poly(I:C) in patient fibroblasts (Fig. 4E and4F).

DISCUSSION

We report on four siblings born to consanguineous parents of Turkish origin affected by a neurodegenerative, multisystemic disease. The patients presented with psychomotor retardation, spasticity, small stature, skin lesions and, most notably, with a triad of stenoses of intracranial vessels, stroke, and glaucoma. Three of these patients had been treated for a multisystemic inflammatory autoimmune process for many years. However, we had been unable to link their symptoms to a known syndrome. Suspecting a hitherto undescribed entity, we performed homozygosity mapping to identify its genetic cause. The genome-wide linkage analysis showed a multipoint LOD score of 3.0 on chromosome 20. A homozygous region spanning 8.1 Mb and harboring more than 170 candidate genes was detected in the four affected siblings. The complete candidate region was then sequenced after enrichment using next generation sequencing technology. The nonsense mutation c.490C>T (p.Arg164X) in exon 4 of the SAMHD1 gene was rapidly identified. The four affected patients were found to be homozygous carriers of this mutation, as expected. SAMHD1 mRNA was expressed in patient fibroblasts, but SAMHD1 protein was absent in patient fibroblasts. Furthermore, patient fibroblasts showed a modulation of the cytokine expression with elevation of basal expression of interleukin-8 (IL8). Interleukin-8 is a cytokine produced by phagocytes and mesenchymal cells exposed to inflammatory stimuli (e.g., interleukin-1 or tumor necrosis factor) and activates neutrophils by inducing chemotaxis, exocytosis and the respiratory burst (Bagiolini and Clark-Lewis 1992). Additionally, stimulated expression of interferon-beta (IFNB1) was reduced. Interferon-beta is a dominant factor in shaping downstream events in the innate and adaptive immune responses subsequent to viral infections [START_REF] Theofilopoulos | Type I interferons (α/ß) in immunity and autoimmunity[END_REF].

While we carried out our study, SAMHD1 was described as a disease-causing gene in AGS [START_REF] Rice | Mutations involved in Aicardi-Goutières syndrome implicate SAMDH1 as regulator of innare immune response[END_REF]. We therefore retrospectively searched for markers supporting the diagnosis of AGS in our patients' medical files and completed diagnostic studies in the two surviving patients. Intracerebral calcifications of the basal ganglia were present on CT in all four patients. Reevaluation of the two surviving patients revealed elevated pterine levels in CSF, while cell counts and IFN-α levels in CSF and IFN-α levels in plasma were normal. Diagnosis of AGS in our four patients was thus confirmed.

AGS had been considered as differential diagnosis in this family because of the presence of intracerebral calcifications. The diagnosis was discarded, however, because the milder phenotype and the triad of stenoses of intracranial vessels, stroke, and glaucoma were considered to be clinically incompatible with the disease. In light of our findings, we conclude that the features present in our patients have to be regarded as an extension of the phenotype of AGS. Interestingly, the p.Arg164X mutation found in our patients was reported most recently in combination with the mutation c.433C>T (p.R145X) in two patients with AGS, who suffered from chronic arthropathy and contractures [START_REF] Dale | Familial Aicardi-Goutières syndrome due to SAMHD1 mutations is associated with chronic arthropathy and contractures[END_REF]. These symptoms were also present to a varying degree in our patients. However, our patients additionally presented with precocious puberty (all patients), calcinosis cutis (patient no. 4) and intracranial "large vessel disease" (patient no. 1, no. 2, and no. 4) not previously reported in AGS patients.

The SAMHD1 gene comprises of 16 exons and encodes a 72.2 kDa protein consisting of 626 amino acid residues. The SAMHD1 protein contains a SAM (sterile alpha motif) domain and a HD (histidine / aspartic acid) domain. SAMHD1 was initially designated as dendritic cell-derived IFN-γ induced protein (DCIP) [START_REF] Li | Identification of human homologue of mouse IFN-γ induced protein from human dendritic cells[END_REF] since it was identified in monocyte-derived dendritic cells as a human homologue of mouse IFN-γ induced protein.

Overall, mutations in SAMHD1 seem to result in a milder phenotype of AGS compared to mutations in TREX1 and RNASEH2 and RNASEH2C [START_REF] Rice | Mutations involved in Aicardi-Goutières syndrome implicate SAMDH1 as regulator of innare immune response[END_REF]. This is supported by our study, as the patients presented here survived into adulthood and one affected patient showed only mild cognitive impairment.

Pathophysiologic mechanisms associated with mutations in SAMHD1 and their relation to mutations in TREX1 or RNASEH2 are not fully understood [START_REF] Crow | Aicardi-Goutières syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity[END_REF]. TREX1 and RNASEH2 are involved in DNA and RNA degradation. Defective TREX1 and RNASEH2 protein complex activities lead to accumulation of DNA and RNA by-products, mimicking viral infection and causing antiviral immune response (Alarcon-Riquelme, 2006;[START_REF] Bhoj | Linking Retroelements to Autoimmunity[END_REF]. SAMHD1 is upregulated in viral infection [START_REF] Préhaud | Virus Infection Switches TLR-3-Positive Human Neurons To Become Strong Producers of Beta Interferon[END_REF][START_REF] Zhao | Inhibition of G1P3 expression found in the differential display study on respiratory syncytial virus infection[END_REF] and is induced by TNF-α [START_REF] Liao | Dendritic cell-derived interferon-γ-induced protein mediates tumor necrosis factor-α stimulation of human lung fibroblasts[END_REF]. Its exact function, however, needs to be further elucidated.

In our patients, SAMHD1 was not expressed when mimicking viral infection in vitro using the Toll-like receptor-3 (TLR3)-agonist Poly(I:C). When stimulated with Poly(I:C), there was also no expression of interferonbeta (IFNB1), a cytokine present in viral infection. Low expression of IFNB1 in fibroblasts of AGS-patients was an unexpected finding. However, this pattern was found in cells obtained from independent patients and was highly reproducible in several independent sets of experiments. Although our data point to a dysregulation in the stress response to inflammatory stimuli, several reasons may explain the lack of IFNB1 up-regulation in fibroblasts in vitro. It is well known that the interferon (IFN) pathway is induced by TLR-dependent as well as by TLRindependent pathways. In our experimental setting only TLR-dependent signalling cascades are activated. Initial TLR-independent activation pathways, however, have been supposed to be highly relevant for autoimmune processes [START_REF] Baccala | TLR-dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity[END_REF]. In addition, an emerging paradigm in innate immunity signalling is that the biological context of the cell, e.g. the intracellular compartimentalization, can influence the outcome of a ligandreceptor interaction, which may differ between the situations in vivo and our experimental setting in vitro [START_REF] Barton | A cell biological view of Toll-like receptor function: regulation through compartmentalization[END_REF]. Last but not least, several groups have identified a pattern of enhanced expression of genes, which are regulated by type I interferons in patients with systemic lupus erythematosus. These findings have shown that mainly myeloid and plasmacytoid dendritic cells, but not fibroblasts, are the relevant sources of interferons in this autoimmune disease [START_REF] Croker | SLE: challenges and candidates in human disease[END_REF]. Thus, high interferon-beta levels found in AGS patients may be due to secondary immune mechanisms during the later course of disease. Therefore, our finding of reduced IFNB1 expression in patient fibroblasts might not reflect the complex pathology of AGS in vivo.

Moreover, basal expression of interleukin-8 (IL8), a potent chemoattractant for neutrophils and a central mediator of vascular inflammation, was increased. This seems to be very specific since other pro-inflammatory molecules like IL-6, ICAM-1 or CXCL9 were not differentially regulated. Interleukin-8 plays a crucial role in initiating atherosclerosis by recruiting monocytes and macrophages to the vessel wall [START_REF] Ito | Inflammatroy cytokines and cardiovascular disease[END_REF] and has been shown, together with IL-6 and RANTES, to be elevated and to correlate with disease activity in patients with Takayasu's arteritis [START_REF] Arnaud | Takayasu's arteritis: An update on physiopathology[END_REF]. Although intracranial stenoses of large cerebral arteries have not been reported previously in AGS, inflammatory microangiopathy is present in a subset of patients with AGS [START_REF] Barth | The neuropathology of Aicari-Goutières syndrome[END_REF]. Neurovascular pathology has also been observed in patients with Retinal Vasculopathy and Cerebral Leukodystrophy (RVCL) and with Hereditary Endotheliopathy, Retinopathy and Nephropathy (HERNS) caused by TREX1 mutations [START_REF] Kavanagh | New roles for the major human 3'-5' exonuclease TREX1 in human disease[END_REF]. We speculate that increased IL-8 levels contribute to the vascular pathology in AGS. However, at this point, it is not clear, whether overproduction of IL-8 in patient fibroblasts is unique to patients with SAMHD1 mutations or whether this is a shared feature in all AGS patients.

In summary, the Arg164X mutation in SAMHD1 was associated with stenoses of intracranial vessels, stroke, and glaucoma, a finding extending the phenotype of AGS. Moreover, our data show a dysregulation of the primary inflammatory stress response in our patients' fibroblasts. It may be speculated that this mechanism may be involved in the vascular pathology of the patients presented here.

Figure 1 .

 1 Figure 1. Radiologic findings in two patients homozygous for the Arg164X mutation in SAMHD1. (A) Patient 3 at age 25: Calcifications (arrows) in the basal ganglia (CT). (B) Patient 4 at age 14: MR angiography showing stenosis of the left distal internal carotid artery (arrow with dashed line) and discontinuation of the left medial cerebral artery (upper full arrow). The A1 segment of the left anterior cerebral artery is not detectable (lower full arrow). On the right, severe stenoses of the internal carotid artery (white arrowhead) and of the proximal part of the medial cerebral artery (double vertical arrow).

Figure 2 .

 2 Figure 2. The SAMHD1 Arg164X mutation cosegregates with the red haplotypes of the family. A haplotype analysis with microsatellite genotype data of the main region of interest further confirmed the locus on chromosome 20. Only the four affected family members were homozygous for the markers in the interval as represented by the surrounding box. The marked individual 1 was chosen for sequencing of the whole region by next generation sequencing technology after DNA enrichment. The stop mutation CGA TGA found in Arg164 of the SAMHD1 gene (GenBank NG_017059.1) was validated by Sanger sequencing. The figure also integrates the red colored mutation as part of the red colored disease haplotypes. Mapping coordinates are given in physical distances according to human NCBI build 37.3.

Figure 3 .

 3 Figure 3. Expression of SAMHD1 protein in human fibroblasts. (A) Western blot with antibody recognizing SAMHD1 protein. Blot was reprobed with β-actin antibody to control for equal loading. (B) Fibroblasts treated with Poly(I:C) and TNF-α for 24 h, lysed and immunoblotted using SAMHD1 antibody. Anti -β-actin antibody was used as a loading control. Co., Control. Poly(I:C), polyinosinic-polycytidylic acid. TNF-α, tumor necrosis factor alpha.

Figure 4 .

 4 Figure 4. Time-course studies of the fibroblasts' response to Poly(I:C). Basal expression of IL8 after 4 (Panel A) and 24 hours (Panel B). Expression of IL8 after stimulation with Poly(I:C) after 4 (Panel C) and 24 hours (Panel D). Expression of IFNB1 after stimulation with Poly(I:C) after 4 (Panel E) and 24 hours (Panel F). The plots show mean ± SEM (n=3). Differences between patients and controls regarding IL8 and IFNB1 expression are statistically significant (p≤0.024, Mann-Whitney test). Control fibroblasts are numbered C2 to C9, patient fibroblasts P2 to P4. Co., Control. IL8, interleukin-8. IFNB1, interferon-beta. Poly(I:C), polyinosinic-polycytidylic acid.

Table 1 .

 1

	F o
	r
	P
	e e r
	R e v i e w

Novel features of Aicardi-Goutières syndrome 3

TABLE 1 Clinical Features of Patients with the Arg164X Mutation in SAMHD1

 1

		Patient 1	Patient 2	Patient 3	Patient 4
	Brain
	Intracerebral Calcifications	+	+	+	+
	Cerebral Atrophy	+	+	-	+
	Stenosis of intracerebral vessels	+	+	+	+
	Stroke	-	-	-	+
	Neurology
	Spacticity	+	+	-	+
	Dystonic posturing	+	+	-	+
	Seizures	-	+	-	-
	Development
	Psychomotor Retardation	+	+	(+)	+
	Eyes
	Glaucoma	+	+	+	+
	Cataract	+	+	-	-
	Skin
	Chilblain-like lesions	-	+	+	+
	Endocrinology
	Hypothyroidism	-	-	+	+
	Precocious Puberty	+	+	+	+
	Musculoskeletal
	Joint contractures	?	+	+	+
	Growth
	Small Stature	+	+	+	+
	Microcephaly	?	+	-	+
	Survival
	Age at death	40 years	29 years	Alive at 25	Alive at 20
	Laboratory Findings
	Elevated Transaminases	?	+	(+)	(+)
	Thrombocytopenia	?	-	-	-
	Lymphocytosis in CSF	+	(+)	-	-
	Interferon-α in CSF	/	/	-	-
	Interferon-α in serum	/	/	-	-
	Pterins in CSF	/	+	+	+
	Data are indicated as follows: +, present; (+), mildly present; -, absent; /, not analysed; ?, data not available.
	Abbreviations: CSF, cerebrospinal fluid

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGMENT

The authors thank Ulrike Botschen and Elisabeth Kirst for expert technical assistance. F.R. and M.d.M. are supported by a grant from the Interdisciplinary Center for Clinical Research, Muenster.