B Sorazu
email: b.sorazu@physics.gla.ac.uk

K A Strain

I S Heng

R Kumar

Violin mode amplitude glitch monitor for the presence of excess noise on the monolithic silica suspensions of GEO 600

Keywords: numbers: 04.80.Nn, 95.55.Ym, 95.75.Kk, 95.75.Pq

Non-Gaussian features of data from gravitational wave detectors are of interest as unpredictable "glitches" limit the sensitivity of searches for many kinds of signal. We consider events due to non-random excitations of the test masses and their suspension fibres. These events could, for example, be related to acoustic emissions in the fibres due to the presence and propagation of cracks or another type of structural perturbation, and they would generate excess noise above the Gaussian background, which matches the level expected due to thermal noise. We look for excess noise in the fundamental violin modes of the monolithic silica suspension fibres of GEO 600. We describe the algorithm used to monitor the violin mode amplitude for glitches, present our results and consider how these may be applied to advanced detectors. The conclusion of our analysis is that no excess noise above what was considered to be thermal noise was observed for several days of h(t) data analysed at the frequency of selected violin modes.

Introduction

The most sensitive gravitational wave detectors (LIGO [START_REF] Sigg | Status of the LIGO detectors[END_REF], VIRGO [START_REF] Acernese | The status of VIRGO[END_REF], GEO 600 [START_REF] Lück | The GEO 600 status[END_REF] and TAMA 300 [START_REF] Takahashi | Status of TAMA 300[END_REF]) employ the technique of laser interferometry to compare the shortterm changes in length of two approximately orthogonal "arms". The arms are defined by the light path between mirrors and a beam-splitter all of which are suspended as pendulums and on vibration isolation systems in vacuum to isolate against external sources of vibration. The mirror (and beam-splitter) suspensions are commonly formed by looping one or more steel wires around the fused silica cylindrical optic. In the case of GEO 600, however, fused silica fibres replace the steel wires. The fibres are welded on to suitably shaped "ears" that are bonded one each side to flats polished to form left and right sides on the otherwise cylindrical substrate [START_REF] Goßler | The suspension systems of the interferometric gravitational-wave detector[END_REF]. The hydroxy-catalysis method is used to form a bond roughly 50 to 100 nm thick [START_REF] Elliffe | Hydroxide-catalysis bonding for stable optical systems[END_REF]. For technical reasons the tops of the fibres in GEO 600 are supported by ears on an "intermediate mass" the same size as the mirror but made from lower quality fused silica.

Although external sources of vibration can be isolated, thermal noise, which can be viewed in terms of the fluctuation-dissipation theorem as resulting from mechanical loss in the materials [START_REF] Gonzalez | Brownian motion of a mass suspended by an anelastic wire[END_REF], cannot be eliminated with components at finite temperature.

Confidential: not for distribution. Submitted to IOP Publishing for peer review 18 May 2010

The main reason for choosing fused silica for the fibres in GEO 600 is that it has low dissipation and hence contributes reduced thermal noise. All of the other components in the suspensions contribute thermal noise, including the mirror substrates, the dielectric coatings on the mirrors, the ears and the bonds between ears and mirror.

In GEO 600 it is believed [START_REF] Smith | Linear projection of technical noise for interferometric gravitational-wave detectors[END_REF] that the noise sources associated with the interferometric measurement of the relative positions of the mirrors (i.e. laser noise, sensing noise, oscillator noise, etc.) exceeds and thus obscures thermal noise over most of the observing band, which extends from 50 Hz to about 3 kHz. An exception is in the close neighbourhood of what we term the "violin modes". These are the transverse modes of vibration of the suspension fibres oscillating between the almost-rigid constraints represented by the mirror and intermediate mass. We could approximate these oscillating thin suspension fibres under considerable stress as oscillators which are in a state of thermal equilibrium with the environment. The motion of these oscillators induced by thermal fluctuations (thermal noise) is Brownian in nature and results in oscillations with random amplitude and phase in every mode of oscillation [START_REF] Bilenko | Measurements of effective noise temperature in fused silica fiber violin modes[END_REF]. In particular the component of the vibration along the sensitive axis of the interferometer leads to motion of the mass that is sensed by the interferometry. This motion is of reduced amplitude, however, as the test mass of many kg reacts to a lesser degree. The randomness associated to the Brownian motion of the interferometric detectors test masses due to thermal noise, shows as changes in the amplitude of the violin modes with a normal (or Gaussian) distribution.

The focus of the current work is to search for events which lead to excitation of the violin modes such that their amplitude variation with time is inconsistent with thermal noise and therefore does not have a Gaussian distribution. This is of interest because such events, evidenced in the form of unexpected "glitches" ‡, limit the sensitivity of searches for several classes of gravitational wave signal. Non-Gaussian events of this type are known to occur in steel suspension metal wires due to stress-release processes [START_REF] Yu | Measurement of excess noise in the suspension fiber for a gravitational wave detector[END_REF][START_REF] Bilenko | Thermal and excess noise in suspension fibres[END_REF][START_REF] Cagnoli | Mechanical shot noise induced by creep in suspension devices[END_REF][START_REF] Beccaria | The creep problem in the VIRGO suspensions: a possible solution using Maraging steel[END_REF]. Similar stress-release processes may happen in some of GEO 600 suspension components, such as fibres and bonds which are permanently under stress. Additionally as the "next generation" detectors, Advanced LIGO [START_REF] Cagnoli | Silica suspension and coating developments for Advanced LIGO[END_REF][START_REF]Advanced LIGO reference design[END_REF] and Advanced Virgo [START_REF] Flaminio | Advanced Virgo white paper[END_REF], are planned to implement fused silica suspension fibres it is of interest to consider whether information can be extracted from the experience of using similar technology in GEO 600.

We are aware of previous work done on a test setup of fused silica fibres alone under tension to observe any non-thermal excitation [START_REF] Gretarsson | Monitoring the thermal and nonthermal excitation of fibers[END_REF][START_REF] Bilenko | The investigation of thermal and non-thermal noises in fused silica fibers for Advanced LIGO suspension[END_REF]. Their conclusion is that no evidence of non-thermal excitation was observed. However their measurements only applied to silica fibres on a down-scaled model of a suspension, meanwhile the work presented here concentrates on a monolithic fused silica suspension for a full scale ground based gravitational wave interferometric detector in long term operation. This paper is structured as follows; first we establish that the observed typical ‡ A glitch is defined, in the context of this text, as a transient signal within the detector recorded output. These transients could be due to gravitation wave sources (short duration violent astronomical events) or most probably to noise transients of instrumental and/or environmental origin due to the great sensitivity of current interferometric gravitational wave detectors.

amplitude of vibration of the mirrors in GEO 600 at the fundamental violin mode resonances, of at least some of the suspension fibres, corresponds to the expected thermal energy excitation. This is achieved with the help of a detailed and accurate model of GEO 600 suspensions using a Finite Element Method (FEM). The FE model provides the relationship between thermal energy, apparent thermal force, and vibration of fibres and mirrors, which were compared to the vibration of the mirrors observed in GEO 600 at the fundamental violin mode frequency. We follow with the description of a computer algorithm designed to monitor the violin mode amplitude for glitches. This algorithm is later applied to several violin modes which are analyzed over timescales of the order of 1 day to look for non-Gaussian events. The result is expressed in terms of the rate, or an upper limit on the rate, of events of a given force. We finish this paper by considering how these results may be applied to advanced detectors.

Finite elements model of GEO 600 suspension

The main objective of this paper is to look for non-Gaussian events affecting GEO 600 suspensions by detecting a non-Gaussian distribution on the changes in the amplitude of the violin modes. However this only would be true if the observed violin mode amplitude is consistent with thermal noise. This would also allow us to extend the results observed in GEO 600 to other systems based in similar suspensions.

In principle we could have taken an analytical approach to show that the observed violin mode amplitude was dominated by an excitation equivalent to the expected thermal energy. However due to the need to represent fibres as relatively complex tapered beams, a finite element approach was favourable. The finite element model approximated a GEO 600 test mass (mirror of weight 5.6 kg) and its four supporting tapered fibres (with length of 28 cm, 10 mm apart, 300 µm at the thinnest section and 3 mm at the stock ends). Fibres of slightly different properties were simulated as no two fibres in GEO 600 are identical and this allowed to break the degeneracy between the fibres in the model to allow the individual outcomes to be extracted. Tapered and un-tapered (uniform cross section of 300 µm) fibres were used for comparison as well as one fibre with a 10% change in its density (as shown on the FE model diagram of GEO 600 suspension shown in figure 1).

The FE model was firstly solved under a static analysis which provided the equilibrium state of the model under gravity which was later used as the initial state for a modal analysis. The modal analysis provided all natural frequencies of the system. The fundamental violin mode of one fibre was investigated. The energy of motion and displacement of the anti-node of the mode (close to the middle of the fibre) and of the mass were compared. This allowed the amplitude of the violin mode, when excited with the expected thermal energy, to be estimated.

The GEO 600 suspension has a high mechanical quality factor (Q). To reduce the settling time after disturbance the fibres were damped by application of polytetrafluoroethane on carefully chosen segments of the total length [START_REF] Goßler | Damping and tuning of the fibre violin modes in monolithic silica suspensions[END_REF]. This reduces the Q of the first few modes to ∼ 10 7 . The fundamental violin mode frequencies in GEO 600 are around 700 Hz, and so a long measurement is needed to resolve the width of resonance. This is not attempted in the current work. Integrating over the narrow resonance, the rms thermal noise level is independent of the Q, as it arises from the usual mode energy of k b T/2 per degree of freedom (here k b is Boltzmann's constant and T is the temperature). Considering that, in first order, only the longitudinal mode is sensed by the detector then a single degree of freedom is used in the estimation of the expected thermal energy (k b T/2 ). We calibrated the displacement and energy results from the FE model by first relating the computed kinetic energy of each mode with the computed displacement of the 5.6 kg test mass. Finally by comparing the expected thermal energy to the computed kinetic energy of the mode it was possible to calculate the thermal noise displacement values shown in Table 1.

Table 1 shows the outcome of the simulations for the four different fibres in the model. The values given by the modal analysis of energy and displacement are relative numbers (the modal analysis includes an arbitrary excitation, but returns results in SI units, which allow scaling to the thermal noise excitation). The total energy refers to the energy of the combined suspension (mass and fibre) and system displacement refers to the antinode of the fundamental violin mode of the fibre oscillation. The total energy of system and mass are given as their kinetic energy at the equilibrium position where the elastic potential energy is minimum (or null). The last column in Notice that the violin modes frequencies obtained from the FE model of the different fibres are a few percent higher than the experimental ones (which range from 627 Hz up to 713 Hz). This discrepancy is due to a lack of precise knowledge of the GEO 600 fibres diameter, which range between 210 and 300 µm depending on the suspension, as well as the simulation fibres not including the damping material applied to GEO 600 fibres. Table 1 shows a spread of no more than 10% in the values of the violin mode amplitude for the different types of simulated fibres.

Finally we need to calculate the experimental violin mode amplitudes as given by the amplitude spectral density of the gravitational wave stream (called h(t)). The typical amplitude, after settling, of a fundamental violin mode in GEO 600 is seen as a strain value which could oscillate by a factor of 2 or more depending on the time and violin mode being measured. For instance for the violin mode at 684.9 Hz the violin mode amplitude lies in the range 7 • 10 -20 to 12 • 10 -20 /

√

Hz rms (for a window bandwidth (FWHM) of 0.012 Hz) (see figure 2 as an example, which is the amplitude spectral density of 30 minutes of h(t) GEO 600 data around the violin mode frequency of 684.9 Hz). This violin mode is associated to the far mirror in the east arm of the detector (identified as MFe) [START_REF] Elliffe | Hydroxide-catalysis bonding for stable optical systems[END_REF], which contributes with an effective length of 600 m for the conversion from strain into displacement. Therefore the amplitude of this violin mode rms oscillates between 4.5 • 10 -18 and 7.9 • 10 -18 m. The same effective length of 600 m is contributed by the beam-splitter although the violin modes associated to the central building mirrors would contribute with an effective length of 1200 m.

The values of the violin mode amplitude given by the FE model are within the experimental range of values and so, given any evidence to the contrary, it is reasonable to assume that the observed violin mode amplitude in GEO 600 is consistent with thermal noise. Therefore any sufficiently large glitch originating as excitation of the suspension fibres, should appear in h(t) as a glitch in the amplitude of the associated violin mode(s). In the following sections we describe the algorithm used to monitor the violin mode amplitude for glitches, present our results, and consider how these may be applied to advanced detectors.

Algorithm to monitor violin mode amplitude and detect glitches

The objective of the algorithm is to monitor and record short-term perturbations in the amplitude of the selected violin modes. The block diagram in figure 3 shows its basic principle of operation. The approach employs a heterodyning, filtering and down-sampling procedure followed by differencing to reveal changes in the complex amplitude of the mode.

The input data to the algorithm is calibrated h(t) data. A check is done that that the detector is operating properly and producing valid, calibrated "science" data throughout the period of time to be analysed.

The h(t) data is queried and divided into overlapping segments of equal length (we name these "heterodyning segments"). In each heterodyning segment the frequency of the violin mode is considered constant. In the analysis presented here the data segment duration was typically chosen to be 5 minutes.

The heterodyning segments overlap in order to avoid the discontinuities being generated at the beginning and end of the data segments due to settling in the low pass filtering stages, without losing data. The overlap was taken to be 80% in the current work. The monitor requires an initial estimate of the frequency of the violin mode (f h0 ). This value can be obtained by plotting the spectrum of, for example, 30 minutes of h(t) data at the centre of the time interval being analysed.

Heterodyning

Each segment of h(t) data is heterodyned and both quadratures are generated by multiplying it by a sine and cosine wave at the appropriate heterodyning frequency (initially f h0 ). This brings the mode frequency and any nearby contaminating spectral features in the data to low frequency, where it is easy to pick out the desired component by low-pass filtering. Both quadratures are needed as the initial phase is unknown and the frequency estimate is in any case imperfect.

Low pass filtering

The next stage in the algorithm is low-pass filtering to isolate the violin mode of interest from the adjacent spectrum peaks. As shown in figure 3, this is done by two down sampling stages. The more selective filter is of finite impulse response (FIR), an equiripple type due to its suitability to specify a minimum stop-band attenuation. The FIR filter is preceded by a down-sampling stage of a 4 th order Butterworth infinite impulse response (IIR) made from two second-order stages to reduce sensitivity to coefficient errors.

Based on an estimate of the required suppression of beats with nearby lines, the FIR filter was chosen to give a minimum stop-band attenuation of 50 dB. A bandpass ripple of 1 dB was allowed as it was considered not to introduce any noticeable perturbations of the amplitude of the violin mode. The FIR filter also required a narrow transition of around 0.1 Hz. Both the IIR and FIR filters had to be adapted for the different violin modes chosen.

Frequency tracker

It was noticed that over a period of several hours the violin mode frequency would shift gradually by a few tens of mHz, most likely due to temperature drifts of the suspension fibres and drift of the pitch and roll alignment [START_REF] Goßler | The suspension systems of the interferometric gravitational-wave detector[END_REF]. The former due to a temperature dependency in the Young's modulus of the fused silica and the later due to the temperature dependency of the Young's modulus of the cantilever springs which support the suspension fibres as part of the seismic isolation scheme. As a consequence our algorithm was made to adjust the heterodyning frequency to keep the heterodyne signal near DC. In order to do this, we took into account that the frequency shift (ω h -ω i ), in magnitude and sign, of the violin mode is encoded in the sine and cosine heterodyne projections (sin h(t) and cos h(t) respectively):

cos h(t) + i sin h(t) = -A 2i e it(ω h -ωi) (1)
The absolute value of the Fourier transform of equation 1 would be a Dirac delta at the frequency (ω h -ω i ), which is what the frequency tracker should monitor.

Glitch power values

The final part of the violin mode amplitude monitor, represented by the last three blocks in figure 3, combines the sine and cosine heterodyne projections and converts that information into the "glitch power" of the violin mode. First we difference the sine and cosine heterodyne projections individually and then find the square of the absolute value of each output. These are summed to obtain the glitch power.

The input h(t) data to the algorithm is given in strain units, so the output glitch power is given in strain squared units (∆L/L) 2 . When considering individual suspensions a displacement is more useful so the output glitch power was converted into glitch displacement squared values by multiplying by the square of the effective arm length of the suspended mass associated to the violin mode being analysed. This effective arm length is of 600 meters for the beam-splitter and far mirrors (these mirrors are identified as MFe (east arm) and MFn (north arm) in GEO 600 and they are the elbow mirrors of GEO 600 folded arms) and of 1200 meters for the arm end mirrors (identified as MCe and MCn in GEO 600 because they are situated in the central building together with the beam-splitter). In the glitch power plots of figures 4 and 5 the length factor was always 600 meters because the corresponding violin modes were associated to the far mirror MFe and the beam-splitter respectively. This algorithm has been successfully applied to several violin modes over a few days of h(t) data. Next we present the result of some of these analyses.

Results and analysis

As with any gravitational wave detector there are glitches of various origin. While the violin mode monitor, through its narrow band response, provides good rejection of background glitches -i.e. those that show up other than just at the violin mode frequency, there is some leakage. In testing the algorithm it was observed that selection of only the quietest stretches of data as judged by other means was a starting point. There is no reason to believe that this selection would lead to bias, provided quite long stretches of data were used (and there has been no further selection after the violin mode analysis).

Selection of the h(t) data and violin modes to be analyzed

The selection of adequate h(t) data segments was done by inspection of the timecentral frequency plots of the glitches detected by HACR §, this is part of the routine data quality monitoring provided for GEO 600 data. The selected data segments were those with the lowest glitch rates and few obvious structures within the glitch concentration, particularly for glitches likely to have significant power around the violin mode frequency. Unfortunately the presence of the violin modes reduces the sensitivity of HACR in the critical band, and so the safe approach was to look for data showing a relative absence of glitches above and below the violin mode band.

GEO 600 provides a data quality channel which gives real time information of the unlocked state of the detector as well as hardware and software maintenance being carried out. We looked for a segment of continuous data (to simplify the analysis) with the detector locked during the entire segment, without any type of maintenance being performed and with low glitch density. Not all the violin modes are suitable for this analysis. Some violin modes are associated to suspended masses with feedback control forces applied to the masses continuously. Control forces are applied during a re-lock of the interferometer to bring it to its operating point (these masses are the central building mirrors; MCe and MCn) and once locked the control forces are applied to keep it locked. These feedback forces would excite the respective violin modes amplitude and any glitches in the control forces would be difficult to distinguish from glitches in the suspension. § Hierarchical Algorithm for Curves and Ridges, it is the event trigger generator used in GEO600 to detect transient-burst events [START_REF] Balasubramanian | Results from the first burst hardware injections performed on GEO 600[END_REF][START_REF] Heng | First steps towards characterizing the hierarchical algorithm for curves and ridges pipeline[END_REF]. HACR produces glitches when an excess power of short time duration is detected. In order to look for excess powers, HACR divides the gravitational wave signal h(t) into highly overlapped milliseconds segments. Every few seconds a spectrogram is calculated with 32 Hz frequency resolution and 7.8 msec. time resolution. Each of these time-frequency resolution squares is called "pixel". HACR groups those adjacent pixels which are statistically different from the background noise into a single glitch.

That is why these violin modes are not used in this analysis. The violin modes that do not change in amplitude during an interferometer re-lock are the ones we name stable and they are clearly visible by looking into ASD (amplitude spectral density) plots of h(t) data before and after a re-lock. The stable modes, from which we selected those to be analysed, were observed to be of relatively stable amplitude and show no sign of a step change during detector re-lock. These modes are associated to the fibres for the beam-splitter and the far mirror suspensions; MTe and MTn.

In GEO there are 12 fibres -4 on each of the two far mirrors and the beam-splitter that are of interest for this work. From these modes we selected a few to analyse based on the nearest neighbour being sufficiently far away in frequency to allow its elimination in the second low-pass filtering stage. It had previously been observed that in some cases the fibres in GEO 600 were of sufficiently non-circular cross section so that their associated violin mode was split in two. Based on this, it is possible that some of the modes selected are actually unresolved pairs, but this should not affect the analysis.

Glitch power histograms of stable violin modes

Putting all the above considerations together 4 violin modes were selected for further analysis at frequencies of 684.9 Hz, 688.725 Hz, 700.425 Hz and 713.77 Hz. There was a gap of at least 0.35 Hz between any mode and its nearest neighbour. The modes were analysed in order of decreasing frequency gap, as it was not clear what the limit would be. For every violin mode being analyzed it was therefore required to readjust the cut-off frequency value of the IIR low pass filter and the stop-band frequency value of the FIR low pass filter in the monitor algorithm, in order to filter out those nearby peaks correctly. The initial heterodyning frequency, overlap between adjacent h(t) data segments and data segment length were also adjusted. Because similar results were obtained for the 4 analysed violin modes, here we only show two sets of results for the modes at 684.9 Hz and 713.77 Hz applied over 22 hours of h(t) data. As an example, figure 2 shows the spectrum around the violin mode frequency at 684.9 Hz, obtained from 30 minutes of data from the data segment analysed. The nearby peak is in this case at a distance of 0.9 Hz. Note that the background noise is relatively white, as it was for all but one of the violin modes investigated.

The results for the mode at 684.9 Hz over 22 hours of continuous h(t) data are shown in figure 4. The top plot is the normalized histogram and the bottom plot is the normalized cumulative histogram (calculated as the number of glitch power counts above the glitch power threshold given in the horizontal axis) of the glitch power values obtained every 0.875 seconds. The vertical axes are normalized by dividing by the length of the data segment in seconds before taking the logarithm. Standard error bars are given and a straight line was fit to the data. The fitting parameters shown include the "goodness of fit" R, and the value very close to unity (0.99996) indicates that the linear fit is valid and so the data follow a Gaussian distribution as expected if the source of glitches is thermal noise. In the cumulative histogram no deviation from a Gaussian distribution is seen to exceed the error bars, therefore no excess noise was observed that would correspond to non-Gaussian glitches. In the normalized histogram we notice a slight deviation from the linear fit at the highest glitch power values, but this occurs because it corresponds to single glitches. For this mode the cut-off frequency of the IIR filter was chosen to be 0.4 Hz, which was also the pass-band frequency of the FIR filter with a stop-band frequency of 0.55 Hz. The equivalent noise bandwidth (ENBW) of the combined filters was 0.343 Hz.

Figure 5 shows the normalized histogram and cumulative histogram, of the glitch power values obtained every 1.17 seconds, for the violin mode at 713.77 Hz. For this mode the cut-off frequency of the IIR filter was chosen to be 0.3 Hz, which was also the pass-band frequency of the FIR filter with a stop-band frequency of 0.415 Hz. The ENBW of the combined filters was 0.272 Hz.

Similar results were observed at different times and for all but one of the violin modes. The mode at 700.425 Hz showed structured background noise of unknown origin (but possibly related to the mains harmonic at 14 times 50 Hz). This resulted in quite strong correlations showing up in the glitch distribution. As the effect was seen only in the case of this particular mode, with the excess background noise, we chose to neglect the data.

Applying the results to advanced interferometers

A consequence of the presence of multiple nearby peaks around each violin mode, is that the bandwidth for monitoring each mode is limited. As the glitch is expected to be of short-duration, the optimum measurement bandwidth, which would be of the order of the glitch bandwidth or the inverse of the glitch duration [START_REF] Pugh | Experiments on and relating to the detection of gravitational radiation[END_REF], is much larger than the available measurement bandwidth. The bandwidth of the algorithm is given by the ENBW of the combined filters applied to each violin mode, which as shown above are very narrow with respect to broadband glitches. Therefore the signal recovered is expected to be almost exactly proportional to the measurement bandwidth.

Before the glitch power histograms, obtained here, can be applied to other detectors, such as Advanced LIGO and Advanced VIRGO, we first need to calibrate them in respect to the sensitivity of the algorithm to the glitches. One way of calibrating the glitch values of the analysis is to compare them with the noise amplitude spectral density target for a detector (sensitivity curves). In this case there would be a factor of the square root of the ENBW of combined filters entering any comparison of glitch size with the noise amplitude spectral density target for a detector. The calibration and application of the results here presented is not the objective of this paper, but both are identified as further work.

Nevertheless, as GEO 600 and advanced detectors share the same silica suspension technology, we make the reasonable assumption that glitches associated with these suspensions will be of broadly similar pulse-shape and duration. Thus the conclusions of the current analysis is applicable to advanced detectors, i.e. that no deviation of glitch distribution for what is expected to be thermal noise has been observed in several days of h(t) data analysed. The glitches might not be identical for GEO 600 and advanced detectors, however, as in the advanced detectors the suspended mirrors are several times heavier. This means that the response to a given (glitch) force is less, but the increased tension in the fibres makes them stiffer, possibly leading to larger initial glitches. Other factors may also influence the difference in the glitches.

Conclusions

The good linear fits of the logarithmic normalized histograms of the power values associated to the glitches on the amplitude of the stable violin modes analysed indicates a Gaussian distribution as expected if the source of glitches is thermal noise. Because no deviation from a Gaussian distribution is seen to exceed the error bars, no excess noise is observed that would correspond to non-Gaussian glitches, at least at a rate of 1 glitch per day.

Figure 1 .

 1 Figure 1. FE model of GEO 600 test mass and the simulated 4 types of supporting fibres. A zoom of the modeled tapered region is also displayed.

Figure 3 .

 3 Figure 3. Block diagram of the violin mode amplitude monitor algorithm. The symbols and components in the block diagram are defined as follows; | | magnitude, phase, ⊗ multiplication, ⊕ addition, D.S. Stage I = down sample stage I, composed of a IIR (infinite impulse response filter) and posterior down sample, D.S. Stage II = down sample stage II, composed of a FIR (finite impulse response filter) and down sample, f h k = heterodyning frequency of the current iteration (the value used for the first iteration is obtained from a high resolution FFT of the h(t) data used in the analysis), ∆f h = change in the heterodyne frequency, including both sign and magnitude, diff = differential value or equivalently x[k + 1] -x[k].

Figure 4 .

 4 Figure 4. Violin mode at 684.9 Hz with 22 hours of continuous h(t) data analysed: (top) Normalized histogram of the glitch power values with error bars. (bottom) Normalized cumulative histogram (calculated as the number of glitch power counts above the glitch power threshold given in the horizontal axis) of the glitch power values with standard error bars. No excess of glitches is noticeable.

Figure 5 .

 5 Figure 5. Violin mode at 713.77 Hz with 22 hours of continuous h(t) data analysed(top) Normalized histogram of the glitch power values with error bars. (bottom) Normalized cumulative histogram (calculated as the number of glitch power counts above the glitch power threshold given in the horizontal axis) of the glitch power values with standard error bars. No excess of glitches is noticeable.

Table 1 .

 1 FE model of GEO 600 suspension analysis results. All columns apart from "Violin mode amplitude rms" are relative numbers from a modal analysis of the model. E k is the kinetic energy given at the equilibrium position where the elastic potential energy is null.

	Fibre design	Mode	System	Total E k Mass E k Violin mode
		frequency displacement	(J)	(J)	amplitude
		(Hz)	(m)			rms (m)
	Un-tapered 1	718.5	2 9 2 .9	1.02 • 10 7	8.68	5.6 • 10 -18
	Un-tapered 2	730.7	2 9 2 .9	1.05 • 10 7	9.0	5.5 • 10 -18
	Un-tapered	753.9	3 0 7 .4	1.12 • 10 7	8.68	5.05 • 10 -18
	(density 2000 kg/m 3 )
	Tapered fibre	783.7	3 0 3 .4	1.21 • 10 7	10.03	5.06 • 10 -18

table 1 ,

 1 which represents the violin mode amplitude rms (maximum displacement of the mass in the longitudinal direction at the fundamental resonance of the suspension fibres), is already calibrated.

Violin mode amplitude glitch monitor for the monolithic suspensions of GEO 600 14 Further work is planned to use the violin mode amplitude algorithm as a realtime monitor of the state of the fused silica suspensions in the way of a potential data quality flag or veto channel.


Patricia ; Alcaide

Begoña Merinero

Ruiz-Sala Pedro

Eva Richard

Rosa Navarrete

Angela Arias

Antonia Ribes

Rafael Artuch

Jaume Campistol

Magdalena Ugarte
email: mugarte@cbm.uam.es

Pedro Ruiz-Sala

Ángela Arias

Pilar Rodríguez-Pombo

DEFINING THE PATHOGENICITY OF THE CREATINE DEFICIENCY SYNDROME

Keywords: Creatine Deficiency Syndrome, GAMT gene, SLC6A8 gene, Metabolic stress, Reactive oxygen species, Mutation Analysis Creatine Deficiency Syndrome, CDS, GAMT, SLC6A8, Metabolic stress, Reactive oxygen species, Mutation Analysis

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

INTRODUCTION

Cell energy demand and supply are balanced and tightly regulated to ensure economic and efficient energy use. Cells with sudden high energy demands, such as neurons, can increase their rate of ATP hydrolysis by several orders of magnitude within seconds of the need arising, yet intracellular ATP levels remain constant. Via reversible phosphorylation by creatine kinase (CK), creatine (Cr) forms part of a high-energy ADP-to-ATP phosphate buffering system [START_REF] Wyss | Creatine and creatinine metabolism[END_REF], allowing the shuttling of high-energy phosphates from the mitochondria to the cytoplasmic sites where they are required. Cr is also a potential neuromodulator or co-transmitter in the central nervous system (CNS), regulating the activity of post-synaptic receptors such as GABA A. (Almeida, et al., 2006b). In mammals, Cr is normally transported through the blood and taken up by the Cr transporter, a 12-transmembrane-spanning protein (known as CT1 or CRTR) that belongs to a superfamily of Na + /Cl --dependent neurotransmitter transporters (SLC6) [START_REF] Snow | Creatine and the creatine transporter: a review[END_REF]. This transporter is encoded by the SLC6A8 gene located at Xq28. Owing to the spontaneous conversion of Cr to creatinine (Crn), which is excreted in the urine, the Cr pool must be maintained via daily nutritional intake and/or de novo synthesis. Endogenous synthesis occurs in the cytosol compartment and involves two enzymes, L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1), which uses arginine and glycine as substrates and yields the intermediate guanidinoacetate (GAA), and S-adenosyl-Lmethionine:N-guanidinoacetate methyltransferase (GAMT, EC 2.1.1.2), which converts GAA to Cr in the presence of S-adenosyl-L-methionine.

Proton magnetic resonance spectroscopy ( 1 H-MRS) has revealed the brains of some patients with mental retardation to have little or no Cr. This discovery led to the identification of three (CDS). Two of these disorders involve problems of Cr biosynthesis caused by deficiencies of the enzymes AGAT (MIM# 602360) [START_REF] Bianchi | Reversible brain creatine deficiency in two sisters with normal blood creatine level[END_REF] and GAMT (MIM# 601240) [START_REF] Stockler | Creatine deficiency in the brain: a new, treatable inborn error of metabolism[END_REF], while the third is an X-linked inherited deficiency of the creatine transporter (CRTR deficiency; MIM# 300036) [START_REF] Salomons | X-linked creatine-transporter gene (SLC6A8) defect: a new creatine-deficiency syndrome[END_REF]. The measurement of GAA and the Cr/Crn ratio in body fluids can be used to identify CDS patients; patients with GAMT deficiency show high GAA concentrations compared to controls, while patients with AGAT deficiency have low levels. Those with a CRTR disorder show normal GAA concentrations but a significantly increased urinary Cr/Crn ratio.

To date, more than 100 patients with a CDS have been reported, mainly with a CRTR or GAMT deficiency (Almeida, et al., 2006a;[START_REF] Dhar | Expanded clinical and molecular spectrum of guanidinoacetate methyltransferase (GAMT) deficiency[END_REF]. Mutation analysis of SLC6A8 and GAMT has recorded the existence at least 50 different nucleotide changes, mostly point mutations or small deletions (HGVS database) (https://portal.biobase-international.com/hgmd/pro/start.php). However, as in other diseases, it has been difficult to establish a clear correlation between the mutation type and the severity of the clinical phenotype.

Patients with CDS suffer motor dysfunctions, developmental delay, mental retardation, epileptic crises, autistic behaviour and speech problems, highlighting the importance of Cr metabolism and transport in the CNS [START_REF] Beard | Synthesis and transport of creatine in the CNS: importance for cerebral functions[END_REF]. Owing to the neurotoxic effects of GAA accumulation in the grey matter [START_REF] Neu | Activation of GABA(A) receptors by guanidinoacetate: a novel pathophysiological mechanism[END_REF], patients with GAMT deficiency also suffer dystonic hyperkinetic movement disorder.

Since CDS can result in a reduction in total cellular ATP, it might also affect intracellular ATP pools via CK-mediated ATP/ADP shuttling. Cr depletion in the brain has been associated with the disruption of neuronal function and changes in mitochondrial structure [START_REF] Andres | Functions and effects of creatine in the central nervous system[END_REF]. Neurodegenerative diseases, such as Alzheimer disease, Parkinson's disease and amyotrophic lateral sclerosis all have the hallmark of mitochondrial dysfunction resulting in ATP depletion and overproduction of mitochondrial reactive oxygen species (ROS). Cr supplementation, however, has a protective antioxidant effect in these conditions, at least in animal models.

Corroborating this possible crosstalk between metabolic stress and the Cr pathway, a new role has been reported for GAMT activity as a p53-inducible modulator of apoptosis, critical for the adaptive response to nutrient stress [START_REF] Ide | GAMT, a p53-Inducible Modulator of Apoptosis, Is Critical for the Adaptive Response to Nutrient Stress[END_REF].

The present study reports the characterisation of nine patients with CDS showing Cr metabolism or transport defects at the biochemical and genetic level, and explores the impact of their specific genotypes on the variables of cellular metabolic stress, including ROS production and apoptosis.

MATERIALS AND METHODS

Patients

The study subjects were nine unrelated Spanish patients with CDS, seven males and two females, ranging from three to 47 years of age. Patients were identified either by their clinical symptoms and abnormal 1 H-MRS scans, or during the investigation of a possible inborn error of metabolism. Following an established flowchart for the differential diagnosis of CDS [START_REF] Almeida | Creatine and guanidinoacetate: diagnostic markers for inborn errors in creatine biosynthesis and transport[END_REF], urine and plasma Cr and GAA levels were analysed by HPLC/MS/MS as a first

step. An increased urine GAA concentration (>150 mmol/mol Crn) and the Cr/Crn ratio (>1.5) F o r P e e r R e v i e w 7 were used as biochemical markers of GAMT and CRTR defects respectively. Six patients were identified as having a possible defect in CRTR activity, and three with a possible defect in GAMT activity. Primary fibroblast cell cultures were used to confirm CRTR and GAMT defects. Creatine transport was measured using [4-14 C]-Cr (0.2 µCi/mL) at physiological concentrations (3-125 µM) [START_REF] Alcaide | A new case of creatine transporter deficiency associated with mild clinical phenotype and a novel mutation in the SLC6A8 gene[END_REF]. After incubation of the cells with [ 13 C 2 ]guanidinoacetate and [ 2 H 3 ]-S-adenosylmethionine, GAMT activity was determined by analysing the final product of the reaction [ 13 C 2 -2 H 3 ]-Cr by HPLC-MS/MS (Verhoeven, et al., 2004).

Clinical assessments were made for all patients by at least one of the authors. Written informed consent to be included in the study was obtained from all subjects (or their legal guardians), or in the case of minors from their parents/legal guardians. Ethical approval for the use of human fibroblasts in the present study was obtained from the Ethics Committee of the Universidad Autónoma de Madrid.

Culture conditions

Skin fibroblasts from patients and controls were cultured according to standard procedures in minimal essential medium (MEM) supplemented with 1% glutamine, 10% foetal calf serum (FCS) and antibiotics in a humidified atmosphere containing 5% CO 2 . Control cell lines (GM03348, GM08333, GM08429, GM08680, GM09503) aged 2-10 years were obtained from the Coriell Institute for Medical Research, NIGMS Human Genetic Cell Repository, USA. Skin fibroblasts from three healthy individuals aged 20-41 years were also included as controls. Most of the cells used in this study were in their 7 th -11 th passage. Unless otherwise stated, assays were performed when the cultures reached at least 80% confluence.

Mutation analysis

Fibroblast cell lines, dried blood spots and/or whole blood were used as sources of DNA and/or RNA. Mutation analysis was performed by direct sequencing of overlapping PCR fragments obtained after amplification of the entire coding sequence of GAMT or SLC6A8. The mutations were further confirmed by sequencing both strands of corresponding exons and their flanking intronic sequences.

Total mRNA was isolated using the Tripure Isolation Reagent (Roche Applied Science, Indianapolis, IN, USA) adhering to the manufacturer's protocol, followed by two-step RT-PCR using oligodT as a primer and employing the SuperScript III First-Strand enzyme (Invitrogen, Carlsbad, CA, USA). Genomic DNA was isolated as previously described [START_REF] John | A Rapid Procedure for Extracting Genomic DNA from Leukocytes[END_REF]. Amplifications of genomic exons were performed using the FastStart kit (Roche Applied Science). Primers to amplify cDNA or genomic sequences were designed according to the cDNA GenBank sequences NM_ 000156.4 for GAMT and NM_005629.3 for SLC6A8, or gene GenBank sequences NT_011255 for GAMT and NT_025965 for SLC6A8. All primer sequences are available upon request. All PCR products were sequenced employing BigDye Terminator v.3.1 mix (Applied Biosystems Foster City, CA, USA) following the manufacturer's protocol, purified in Performa V3 96-well short plates (EdgeBio, Gaitherburg, MD, USA), and analysed by capillary electrophoresis using an ABI Prism 3700 Genetic Analyzer (Applied Biosystems).

DNA mutation numbering was based on the cDNA reference sequences taking nucleotide +1 as the A of the ATG translation initiation codon. The nomenclature used follows the recommendation of the Human Genome Variation Society (http://www.hgvs.org./mutnomen/) [START_REF] Dunnen | Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion[END_REF]. In silico analysis Conservation of the mutated residues was assessed by alignment of orthologous and human protein sequences using ClustalW software (http://www.ebi.ac.uk/clustalw/) [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF]. The putatively damaging effects of the predicted amino acid substitutions were assessed using the PolyPhen programme (http://genetics.bwh.harvard.edu/pph/) [START_REF] Ramensky | Human non-synonymous SNPs: server and survey[END_REF]; the results are given as "benign", "possibly damaging", "probably damaging" or "unknown".

The bioinformatic analysis of potential splicing aberrations was performed using Human Splicing Finder (HSF) v.2.4 software: http://www.umd.be/HSF/ (web-based) [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF].

Real-time PCR quantification

SLC6A8 and GAMT mRNA expression levels were assessed in patient and control cell lines by performing qRT-PCR, using ProbeFinder software (Roche Applied Science) for the optimal design of the real-time PCR primers, and the Universal Probe Library (Roche Applied Sciences) for probe selection. Total RNA obtained as described above was retro-transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems) and PCR-amplified using the Fast-Start Taqman Probe Master kit (Roche Applied Science). The GAPDH mRNA expression level was used as an endogenous control. Real-time PCR and analysis were performed in an ABIPRISM 7900HT Genetic Analyzer (Applied Biosystems) using the SDS2.1 programme. RNA integrity was analysed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Relative quantification of gene expression was analysed by the comparative threshold cycle (Ct) method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF][START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF]; ∆C T was calculated as the difference in C T between the target gene and the housekeeping gene for each sample. The following formula was used to calculate the relative amount of transcripts in patients with respect to controls: ∆∆C T = ∆C T (patient) -∆C T (control) for each gene. The fold-change for each gene in each patient relative to the control was calculated as 2 -∆∆CT .

ROS and apoptosis assays

For ROS and apoptosis assays, skin fibroblasts from three controls and the nine CDS patients were cultured as previously described [START_REF] Richard | Methylmalonic acidaemia leads to increased production of reactive oxygen species and induction of apoptosis through the mitochondrial/caspase pathway[END_REF]. The cells were detached by trypsinisation for 1 min, which was immediately stopped by adding complete medium. ROS levels (H 2 O 2 ) were monitored using the H 2 DCFDA fluorescence probe (2,7dichlorodihydrofluorescein diacetate; Molecular Probes, Inc., Eugene, OR, USA) (5 µM) by flow cytometry after 30 min of incubation at 37ºC in the dark.

Apoptotic cells were identified and quantified by flow cytometry using the Annexin V-DY634 apoptosis detection kit (Immunostep Research, Salamanca, Spain). Fibroblasts harvested by trypsinisation as mentioned above were incubated with 2.5 µL of Annexin V-DY634 for 15 min at room temperature in the dark.

Propidium iodide (0.001%; SIGMA, St Louis, MO, USA) was used to detect dead cells.

Samples were analysed using a FACSCalibour apparatus with two solid-state lasers (488 and 633 nm) (Becton Dickinson, Franklin Lakes, NJ). A minimum of 10,000 individual events were recorded. Data were acquired and analysed using Cell Quest-Pro (Becton Dickinson) and FlowJo software (FlowJo, Ashland, OR, USA) respectively.

Flow cytometric analysis of the cell cycle and nuclear DNA content

Cells were grown to a density of 80% and the culture medium replaced by MEM with 0.5% FCS for synchronisation over 48 h. The cells were then transferred back to MEM +10% FCS for 24 h followed by harvesting and counting. 1.5 x 10 6 cells were fixed in 70% ethanol for at least 18 h at -20ºC, and treated with PI/RNase Staining Buffer (BD Pharmingen, San Diego, CA, USA) for 30 min at 37ºC in the dark [START_REF] Kues | Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors[END_REF][START_REF] Nishikiori | Influence of chlorine dioxide on cell death and cell cycle of human gingival fibroblasts[END_REF]. The DNA content was analysed by flow cytometry using a BD FACSCanto II flow cytometer (BSBioscience, San Jose, CA). At least 10,000 events were acquired per sample. Data were acquired using FACSDiva software and the relative percentages of cells in the sub-G0/G1 (apoptotic cells), G0/G1, S and G2/M stages determined using ModFit LT v.2.0 TM software (Verity Software House Inc., Topsham, ME, USA).

Western blot analysis

A total of 4 x 10 5 fibroblast cells of each line were placed in 6-well plates 24 h prior to protein analysis. Cells were lysed with 50 µl/well of ice-cold lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 mM Tris-HCl pH 7.5, protease and phosphatase inhibitor cocktail tablets [Roche Applied Science]). After centrifugation, the protein content of the supernatants was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using BSA as a standard. Samples were loaded onto 4-12% NuPAGE Novex Bis-Tris minigels (Invitrogen, Carlsbad, CA, USA) and blotted onto nitrocellulose membranes using the iBlot Dry Blotting system (Invitrogen). Poinceau staining was used to verify equal protein loads. Western blotting 12 was performed using the phospho-p38 MAP kinase (Thr180/Tyr182) polyclonal antibody (Cell Signaling Technology, Inc, Danvers, MA, USA) and p38 polyclonal antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA). The secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (Santa Cruz Biotechnology); these were detected using the Enhanced Chemiluminescence System (GE Healthcare, Berkshire, UK).

Protein quantification was performed using a calibrated densitometer GS-800 (Bio-Rad Laboratories).

Fibroblast creatine content

Confluent cells from CDS patients and controls were harvested by trypsinisation, pelleted by centrifugation and stored at -80ºC. Cell pellets were resuspended in 0.2 mL of PBS, frozen and thawed three times in liquid nitrogen, and centrifuged (14,500xg for 5 min). The supernatant was used for Cr quantification by HPLC/MS/MS, employing stable isotope-labelled Cr as an internal standard, and for protein determination by the Lowry method. The Cr content was expressed in pmol/µg protein.

Statistical analysis

ROS, apoptosis, cell cycle stage and nuclear DNA contents were determined in at least three independent experiments, each performed in triplicate. Values were expressed as means ± SD unless otherwise stated. All p values were two-tailed. Significance was set at p<0.05.

RESULTS

Biochemical data and mutation analysis of CDS patients

The relevant biochemical and clinical information of the patients with a defect in either SLC6A8 (CRTR patients) or GAMT (GAMT patients) is included as Supporting Information (Supp. Table S1 and Supp. Table S2).

Eleven nucleotide sequence variations were identified that presumably lead to loss of function: six in SLC6A8 and five in GAMT. Of these, seven have been previously (and specifically) reported in Spanish patients [START_REF] Alcaide | A new case of creatine transporter deficiency associated with mild clinical phenotype and a novel mutation in the SLC6A8 gene[END_REF]Fons, et al., 2009;Item, et al., 2004;Sempere, et al., 2009a;Sempere, et al., 2009b). Table 1 shows their distributions in the two genes. The rest correspond to allelic variants previously described in other populations [START_REF] Bizzi | X-linked creatine deficiency syndrome: a novel mutation in creatine transporter gene SLC6A8[END_REF][START_REF] Clark | X-linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology[END_REF]Item, et al., 2004). Most of these changes affect residues highly conserved between the corresponding human proteins and their homologues, as determined by BLAST analysis and comparisons made against sequences available in the NCBI nucleotide database (www.ncbi.nlm.nih.gov) using ClustalW software. Table 1 shows that all missense mutations were predicted to be "damaging" in PolyPhen analysis.

In SLC6A8, the distribution of nucleotide changes included three nucleotide substitutions (a nonsense change and two missense mutations) and three in-frame small deletions. Most of the changes identified affected residues in the transmembrane domains of the protein.

Given the hemizygous condition of the SLC6A8 mutants, Cr transport was directly measured at physiological substrate concentrations in patient fibroblasts to characterise the effect of the mutations. All patient fibroblasts showed a significant reduction in Cr uptake compared to control cells (<20%) (data not shown). In GAMT, the mutant alleles involved two missense and two nonsense changes (one of each in heterozygous fashion in patients P7 and P9), and one small deletion in a homozygous fashion in patient P8. Three of these changes have previously been identified in the Spanish population.

The remaining mutations corresponded to the p.W20S mutation, which appears to be very common in Portugal [START_REF] Almeida | A prevalent pathogenic GAMT mutation (c.59G>C) in Portugal[END_REF], and to p.W174X, which has been described in other populations (Item, et al., 2004).

Genetic analysis of the available family members (Supp. Table S1 and Supp. Table S2) showed that GAMT changes co-segregated in the respective families. However, controversial data were obtained for the SLC6A8 gene; co-segregation could only be demonstrated for the change c.1079_1081delTCT. Similar results have been previously reported for other SLC6A8 mutants.

Although the most plausible explanation would be that these mutations arose de novo as described in [START_REF] Battini | Mental retardation and verbal dyspraxia in a new patient with de novo creatine transporter (SLC6A8) mutation[END_REF], somatic mosaicism cannot be excluded [START_REF] Betsalel | Detection of low-level somatic and germline mosaicism by denaturing high-performance liquid chromatography in a EURO-MRX family with SLC6A8 deficiency[END_REF].

As the mutational spectrum identified included several changes leading to premature termination codons (PTCs) capable of inducing nonsense-mediated mRNA decay, the expression profiles of SLC6A8 and GAMT were examined (Fig. 1). The mRNA levels (quantified by qRT-PCR) of the SLC6A8 alleles and c.145delT GAMT allele appearing in homozygous fashion in patient P8 were compared to those of the control fibroblast cell lines (10-40 years old). Cell lines from GAMT patients bearing the nonsense changes in heterozygous fashion were also included. The results showed a clear decay of the PTC-bearing alleles analysed; this decay, however, was rescued when cells were treated with the translation inhibitor emetine (data not shown). Interestingly, the relative abundance of the SLC6A8 allele carrying the nucleotide variation c.1079_1081delTCT (P3) was also significantly reduced compared to controls. Some exonic mutations may alter normal mRNA processing and lead to the synthesis of aberrant mRNA, which may or may not be

Metabolic stress variables

ROS are products of normal metabolism, and depending on their concentration can be beneficial or harmful to cells [START_REF] Circu | Reactive oxygen species, cellular redox systems, and apoptosis[END_REF]. A number of pathologies can result from oxidative stress-induced apoptosis following increases in ROS [START_REF] Ma | Transcriptional responses to oxidative stress: pathological and toxicological implications[END_REF]. To explore the impact of the CDS mutant alleles on metabolic stress variables, intracellular ROS contents, apoptosis levels and the progression of the cell cycle were examined in all nine CDS cell lines.

Reactive oxygen species

The comparison of patient and control fibroblasts showed those of patients P1 and P3 (CRTR patients), and P7 and P9 (GAMT patients), to have significantly increased ROS levels (1.5-2.4fold, with those of P3 the highest) (Fig. 2).

The activation of a protein belonging to the mitogen-activated protein kinase (MAPK) superfamily, p38 kinase, was also analysed by immunoblotting to compare its activation with the ROS levels detected in CDS patients. The expression pattern of the phosphorylated p38 protein was individually normalised by the protein expression of total p38 protein, which includes both 16 the phosphorylated and non-phosphorylated forms. The expression of phospho-p38 was most strongly detected in the cell lines of the CRTR patients (P1 and P3), i.e., those with the highest ROS levels (Fig. 3). Quantitative densitometry showed a >10-fold higher expression for phospho-p38 in the P1 and P3 cell lines compared to controls (p<0.05). The CRTR cell lines with the lowest ROS contents (i.e., cells from P2, P4 and P6) showed the weakest activation of this kinase (below or equal to that seen in the controls). The results obtained for p38 protein activation correlated with the ROS levels in the CRTR patients' cell lines; however there was no clear correlation between phospho-p38 expression and ROS levels in the GAMT cell lines.

Apoptosis in CDS cell lines

To address the possibility that elevated ROS levels might induce apoptosis in the patient cell lines, the percentage of annexin V-positive cells was investigated by flow cytometry using dual staining with annexin V and PI. This method identifies the apoptotic mode of cell death: annexin V-positive/PI-negative cells were considered early apoptotic cells and annexin V-positive/PIpositive as late apoptotic or necrotic cells. An increase in annexin V-positive/PI-negative (1.3-2fold) and annexin V-positive/PI-positive (1.25-2.5-fold) cells was seen for patients P1, P3, P7 and P9 (Fig. 4). The greatest binding of annexin V was seen in cells from patients P3 and P9.

These results indicate that fibroblasts from CDS patients with increased ROS levels also show an increase in both early and late apoptosis. DNA stainability (sub-G0/G1 peak) [START_REF] Nicoletti | A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[END_REF][START_REF] Umansky | In vivo DNA degradation in thymocytes of gamma-irradiated or hydrocortisone-treated rats[END_REF]. Figure 5 shows that most of the CDS cell lines had an altered cell cycle distribution pattern compared to the control lines (including fibroblasts from adults). Cell lines from patients P1, P3, P7 and P9 showed a very significant percentage of cells at the sub-G0/G1 stage, as seen from their reduced DNA content (less fluorescence and diminished forward light scatter relative to the peak for cells at G1 [14±2 for P1, 18 ±11 for P3, 15±0.9 for P7 and 18±2.7 for P9], indicating apoptosis. A significant accumulation of cells in the G2/M phase was also detected, delaying the transition to G1 in most of the patient cell lines.

Progression of the cell cycle

Intracellular creatine content

To correlate the apparent different behaviours of the patient cell lines in the context of metabolic stress with their capacities to maintain Cr homeostasis, an MS-MS method was used to measure the intracellular Cr levels in fibroblasts grown under standard conditions. Table 2 shows that all CRTR cell lines, irrespective of their genotype, suffered a severe reduction in their intracellular Cr content. However, in all the GAMT cell strains studied, Cr levels were similar to control values, indicating that Cr was efficiently taken up from the culture medium. A total of 11 variants (four missense mutations, three nonsense mutations, three in-frame deletions and 1 frameshift change) were identified in the present cohort of six CRTR and three GAMT patients. The supposed pathogenicity of the missense changes and the small in-frame deletions was based on the conservation of the mutated residue across orthologous proteins, and on in silico predictions. Most of mutations found affected conserved residues, and all belong to stretches of conserved amino acid. The PolyPhen bioinformatic prediction suggested all the missense changes analysed to be potentially harmful to the function of the corresponding proteins. Special importance was associated with the GAMT p.T136M change, which was predicted to lead to a change in the hydrophobicity of a residue at the core of the protein.

DISCUSSION

Genetic variability

However, its pathogenicity needs to be tested in overexpression studies [START_REF] Almeida | A prevalent pathogenic GAMT mutation (c.59G>C) in Portugal[END_REF]Rosenberg, et al., 2007). The frameshift and nonsense variants were highly likely to be pathogenic since they result in dysfunctional and perhaps harmful proteins. Moreover, the drastic reduction of SLC6A8 or GAMT transcripts in the CRTR and GAMT cell lines carrying nonsense or frameshift changes was consistent with the eliciting of nonsense-mediated mRNA decay. An exception to the reading frame rule was obtained for the small in-frame deletion c.1079_1081delTCT which provoked a significant reduction in SLC6A8 mRNA in the P3 cell line. Although the bioinformatic prediction suggested this could be explained by a change in the splicing control sequences, the experimental data reflected mRNA instability unassociated with the generation of aberrant transcripts containing PTCs.

Correlating genotypes with phenotypes is not straightforward in CDS. Patients with genetic CDS usually show important neurological symptoms, but the lack of standardised criteria for classifying the severity of disease limits any associations that might be made. In the present 19 cohort, the mildest disease presentation was seen in P4 patient, who carried p.A404V, the only change affecting a non-conserved residue of the CRTR protein [START_REF] Alcaide | A new case of creatine transporter deficiency associated with mild clinical phenotype and a novel mutation in the SLC6A8 gene[END_REF].

Creatine deficiency and metabolic stress

There is increasing evidence that oxidative stress and apoptosis are closely associated phenomena that might explain the neuropathological aspects of several metabolic and neurodegenerative diseases. At normal concentrations, ROS play a role in signal transduction, including that involved in proliferation. However, an imbalance between ROS generation and the capacity of antioxidants to neutralise them can result in the disruption of the cellular redox status, leading to oxidative stress [START_REF] Circu | Reactive oxygen species, cellular redox systems, and apoptosis[END_REF].

The present results show, for the first time, an overproduction of ROS in two CRTR (P1 and P3) and two GAMT (P7 and P9) patient fibroblast cell lines. These cell lines also showed increased apoptotic rates, measured as the percentage of annexin V-positive cells. This suggests that elevated ROS levels might induce apoptosis in CDS cell lines, as previously suggested for other conditions [START_REF] Benhar | ROS, stress-activated kinases and stress signaling in cancer[END_REF][START_REF] Kim | Oxidative stress is linked to ERK1/2-p16 signaling-mediated growth defect in ATM-deficient astrocytes[END_REF][START_REF] Ma | Transcriptional responses to oxidative stress: pathological and toxicological implications[END_REF].

A loss of the redox control of the cell cycle could lead to aberrant proliferation [START_REF] Sarsour | Redox control of the cell cycle in health and disease[END_REF]. Cell cycle progression is regulated by checkpoint controls that protect the integrity of the genome [START_REF] Hartwell | Checkpoints: controls that ensure the order of cell cycle events[END_REF]. If cycling cells are damaged they usually have the capacity to pause temporarily at G1, S, or G2, repair the damage (if possible), and then re-enter the cell cycle. When damage is severe, however, cells may undergo programmed apoptosis [START_REF] Shackelford | Oxidative stress and cell cycle checkpoint function[END_REF]. In the present work, a significant percentage of cells were found to be at the sub-G0/G1 stage in the cells of patients P1 and P3 (CRTR), and in those of P7 and P9 Replicative cell division is an energetically demanding process. To successfully undergo it, cells must have an adequate supply of essential nutrients to support both ATP production and macromolecule synthesis [START_REF] Jones | AMP-activated protein kinase induces a p53-dependent metabolic checkpoint[END_REF]. In the present work it was first hypothesized that the altered cell cycling pattern observed in the CDS strains might reflect energy depletion as a result of the reduced Cr content. However, when intracellular Cr levels were measured, only cells from CRTR patients showed a drastic reduction in Cr (Cr concentrations were normal in the GAMT cells). In addition, intracellular Cr levels did not always correlate well with the cellular stress phenotype in the CRTR patients -P2, P5 and P6 manifested a mild (or no) cellular stress phenotype. These data strongly support that Cr uptake is the main pathway involved in maintaining Cr homeostasis in fibroblasts, and underscore the role of Cr metabolism in the response to cellular stress. Indeed, ROS damage observed in the P7 and P9 GAMT cells cannot be explained by reduced high energy phosphate reserves since the intracellular Cr levels were in the same range as the controls. Previous research in animals showed that GAA inhibits Na + -K + -ATPase activity, which might be mediated by nitric oxide formation [START_REF] Zugno | Evaluation of the mechanism underlying the inhibitory effect of guanidinoacetate on brain Na+, K+-ATPase activity[END_REF].

Reduced brain antioxidant capacity related to GAA accumulation is also reported in the rat striatum, indicating the possible involvement of oxidative injury, offering an explanation for the pathogenic mechanism of brain damage in patients with GAMT deficiency [START_REF] Zugno | Guanidinoacetate decreases antioxidant defenses and total protein sulfhydryl content in striatum of rats[END_REF]. 21 lines that showed the highest ROS and apoptosis levels. However, the activation of this protein was not detected in the GAMT patient fibroblasts that showed an increase in ROS and apoptosis. These promising results in GAMT cell lines will be followed up with further studies focusing on the components of the signalling pathways that culminate in apoptosis in this metabolic disorder.

ROS levels and apoptosis rates were particularly high in patients P1 and P3, who showed a severe form of CRTR disease and a drastic reduction in the abundance of the SLC6A8 transcript.

It is possible that ROS production exacerbates, helps perpetuate, or causes the neurological phenotype of CRTR patients. Moreover, in patient P4, who had the mildest form of the disease and the highest intracellular Cr content, neither increased ROS nor any other signs of metabolic stress could be detected. However, while ROS production has been reported to possibly affect the nervous system, a complex interplay between GAMT expression and the tumour-suppressor p53 protein has been recently proposed [START_REF] Ide | GAMT, a p53-Inducible Modulator of Apoptosis, Is Critical for the Adaptive Response to Nutrient Stress[END_REF]. In this new link, GAMT appears as a p53-inducible modulator of apoptosis and a key downstream effector of adaptive response to genotoxic and nutrient stress [START_REF] Ide | GAMT, a p53-Inducible Modulator of Apoptosis, Is Critical for the Adaptive Response to Nutrient Stress[END_REF][START_REF] Vousden | Alternative fuel--another role for p53 in the regulation of metabolism[END_REF]. CDS cells could be used as a model to explore the metabolic functions of p53 and to identify critical points in these pathways that might serve as targets for therapeutics interventions.

In summary, this study is the first in which increased metabolic stress measured as ROS production, apoptosis and/or aberrant cell proliferation has been identified in selected CRTRand GAMT-deficient fibroblast cell lines (this information is summarised in Supp. Table S3). In addition, a reduction in high energy phosphate reserves appears as a plausible mechanism for explaining the damage observed in selected CRTR fibroblasts. The existence of a dense genetic interaction network makes genotype-phenotype correlations difficult to identify (as for most hereditary diseases) (Dipple and McCabe, 2000a;Dipple and McCabe, 2000b;[START_REF] Lehner | Modelling genotype-phenotype relationships and human disease with genetic interaction networks[END_REF].

  Cr metabolism, collectively known as creatine deficiency syndrome

  proposed crosstalk to occur between metabolic stress and the Cr pathway.

  modelling can help to assess the potential effect of such variants on splicing. In this work, HSF software was used to predict the effects of mutations on splicing signals or to identify splicing motifs. According to the HSF prediction, the change c.1079_1081delTCT disrupts a low score potential acceptor splice site (score: 66.33→20.51) and alters enhancer motifs recognised by SC35, SRp40 and SRp55 proteins, either breaking the site or creating a new mutant motif. However, no aberrant transcript could be identified after sequencing.

  reduced DNA contents and can therefore be recognized as cells with low

  . The significant inhibition of G2M/G1 transition, identified in most of the CDS cell lines, indicates the position of a possible checkpoint.

  p38 MAPK was found to have a different activation pattern in the CRTR and GAMT cell lines, suggesting that the apoptotic response to genotoxic damage in these cells might be triggered by different signalling pathways. In fact, the activation of p38 MAPK, commonly related to stress response signals such as oxidative damage, was particularly noticeable in the CRTR patient cell

  the present work, an association was seen between genotypes and metabolic stress phenotypes, especially in certain CRTR patients. The present data support the potential of reducing intracellular ROS and oxidative stress-induced apoptosis as a strategy for the treatment of these CDS.

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Analysis of intracellular ROS content in CDS patient cell lines. Results are expressed

Figure 3 .Figure 4 .

 34 Figure 3. Analysis of phosphorylated p38 stress-activated protein kinase expression in patient

Figure 5 .

 5 Figure 5. Analysis of cell cycle distribution in CDS patient and control fibroblasts by flow

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1: Quantification of SLC6A8 and GAMT mRNA by qRT-PCR in CDS patient and control fibroblasts. (A) mRNA level quantification in GAMT patient cell lines. (B) mRNA level quantification in CRTR patient cell lines. The X axis represents cells according to their type of mutation; the Y axis represents the results of the relative quantification (RQ) of the patient cells, where RQ=2-∆∆CT . The following formula was used to calculate the relative amount of transcripts in patient cells with respect to controls: ∆∆CT = ∆CT patient -∆CT control. ∆CT was calculated as the difference in CT between the target gene and the housekeeping genes for each patient sample. Data represent the mean ± SD of three independent experiments (*p<0.05; **p<0.01). P: patient. 268x190mm (200 x 200 DPI)

  -MRS: reduced creatine signal GenBank reference sequences: NM_005629.3 for SLC6A8; NM_000156.4 for GAMT NV: Normal value; y: years F: Genetic analysis of family members performed 1(Item, et al., 2004) 2(Sempere, et al., 2009b) Alcaide et al., Human Mutation 3Supp. Table S3. Summary of test results in CDS patient cell linesGenBank reference sequences: NM_005629.3 for SLC6A8; NM_000156.4 for GAMT NS: not studied. The data represent the mean ± SD of three independent experiments. * p<0.05; ** p<0.01; *** p<0.001

Table 2 . Intracellular creatine content in fibroblasts from CDS patients and controls

 2

	CELL LINE	DEFECT	CREATINE
	P1	CRTR	0.60±0.08
	P2	CRTR	0.55±0.07
	P3	CRTR	0.85±0.35
	P4	CRTR	2.30±0.001
	P5	CRTR	0.60±0.14
	P6	CRTR	0.85±0.07
	P7	GAMT	10.0±0.9
	P8	GAMT	20.8±4.2
	P9	GAMT	NS
	Controls (n:9)		15.3±6.8
	Range		6.8-31.9
	Creatine content expressed as pmol/µg protein
	Values are mean ± SD of at least two biological replicas measured in duplicated.
	NS: not studied

Supp. Table S2. Biochemical and genetic characterisation of GAMT patients

  Alcaide et al., Human Mutation 2

	PATIENT DEFECT	GENOTYPE	GAA	GAMT ACTIVITY	CLINICAL HISTORY
				(mmol/mol Creat)	(pmol/h.mg prot)
				NV (<11y): 13-139	NV:100-195
				NV (>12y): 12-100
	7 1	GAMT	c.[59G>C]+[521G>A]	228	2.8	Female 23y. Severe mental retardation, refractory
			p.[W20S]+[W174X]			epilepsy, autistic behaviour
						Brain 1 H-MRS: reduced creatine signal
	8 F, 2	GAMT	c.[145delT]+[145delT]	201	3.7	Female 12y. Severe mental retardation, refractory
			p.[Y49fs]+[Y49fs]			epilepsy, autistic behaviour
						Brain 1 H-MRS: reduced creatine signal
	9 F, 2	GAMT	c.[316C>T]+[407C>T]
			p.[Q106X]+[T136M]

John Wiley & Sons, Inc. Human Mutation

ACKNOWLEDGMENTS

This work was funded by grants from Ministerio de Sanidad (FIS PI051180, PI052080 and PI051200). The authors thank Ascensión Sánchez for excellent technical assistance with cell culturing. CIBERER is an initiative of the ISCIII (MICIN, Spain). The institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular "Severo Ochoa" is gratefully acknowledged.

John Wiley & Sons, Inc. Human Mutation John Wiley & Sons, Inc. Human Mutation John Wiley & Sons, Inc. Human Mutation John Wiley & Sons, Inc. Human Mutation John Wiley & Sons, Inc. Human Mutation

 (Fons, et al., 2009)2 (Sempere, et al., 2009a) 3 [START_REF] Alcaide | A new case of creatine transporter deficiency associated with mild clinical phenotype and a novel mutation in the SLC6A8 gene[END_REF]