T A R Hansen

G J Colsters

C M Van De Sanden

R Engeln
email: r.engeln@tue.nl

Investigating the flow dynamics and chemistry of an expanding thermal plasma through CH(A-X) emission spectra

Keywords: numbers: 52.25.Fi, 52.40.Hf, 52.70.Kz, 52.77.Bn

The gas flow in a linear plasma reactor and the plasma chemistry during hydrogenated amorphous carbon and graphite etching are investigated via time and spatially resolved measurements of the ion density and CH emission. A convolution of the ion and hydrocarbon density shows the importance of charge transfer in the plasma chemistry which ultimately yields the CH emission. The spatially resolved measurements clearly visualize the plasma expansion in the reactor and its deflection on a substrate. A stagnation zone is furthermore formed in front of a substrate when placed inside an expanding thermal plasma. An increased ion density further upstream from the substrate is attributed to a reorganisation of the gas recirculation cells in the background of the reactor. The movement of a shutter in and out of the plasma expansion likewise affects these recirculation cells. This movement consequently redirects the energy flow to and from the background, as is deduced from the variation in rotational temperature of the CH radical.

Introduction

The next generation experimental fusion reactor, called iter, is currently under construction in the south of France [1]. Its main purpose is to prove the viability of controlled fusion processes as a power source by achieving a tenfold power multiplication [START_REF] Kleyn | Plasma-surface interaction in iter[END_REF]. Such controlled fusion processes require core plasma temperatures on the order of 10 keV. Even though temperatures drop significantly by the time the plasma reaches the wall contact points inside the divertor area, wall erosion remains one of the unresolved issues in fusion research. This is particularly the case under the extreme plasma conditions anticipated in iter. Wall components inside iter's divertor will face an expected ion and energy flux of up to 10 24 m -2 s -1 and 5 to 10 MW m -2 , respectively, for a duration of 500 s or longer [START_REF] Kleyn | Plasma-surface interaction in iter[END_REF]. How well various materials, such as carbon or tungsten, will withstand such plasma conditions is a topic of active research. In particularly, the ion energy range between thermal temperatures and 10 eV under high flux conditions is largely unexplored [START_REF] Roth | Status of knowledge of chemical erosion of carbon and critical issues for extrapolation to iter[END_REF].

Wall materials for experimental fusion reactors are typically first tested in linear plasma reactors [START_REF] Goebel | Plasma surface interaction experimental facility (pisces) for materials and edge physics studies[END_REF][START_REF] Balden | New weight-loss measurements of the chemical erosion yields of carbon materials under hydrogen ion bombardment[END_REF][START_REF] Kornejew | Chemical erosion of cfc at high ion flux densities[END_REF][START_REF] Roth | Flux dependence of carbon chemical erosion by deuterium ions[END_REF][START_REF] Xie | Deposition of a-c:h films on uhmwpe substrate and its wear-resistance[END_REF]. Dedicated plasma reactors provide greater experimental flexibility and can test new materials in anticipation of next generation fusion reactors.

Another advantage of linear plasma reactors is the spatial separation between the plasma source and target. This separation allows for high particle fluxes in addition to low plasma temperatures near the substrate. Several reactors dedicated to the study of nuclear materials, such as psi-i, psi-ii and pisces, have reached ion fluxes above 10 22 m -2 s -1 [START_REF] Goebel | Plasma surface interaction experimental facility (pisces) for materials and edge physics studies[END_REF][START_REF] Balden | New weight-loss measurements of the chemical erosion yields of carbon materials under hydrogen ion bombardment[END_REF][START_REF] Kornejew | Chemical erosion of cfc at high ion flux densities[END_REF]. To test wall materials in even more extreme conditions, as those expected in iter, a new facility called Magnum-PSI was recently constructed in The Netherlands [START_REF] Kleyn | Plasma-surface interaction in iter[END_REF][START_REF] Van Eck | Modeling and experiments on differential pumping in linear plasma generators operating at high gas flows[END_REF][START_REF] Westerhout | Psi research in the iter divertor parameter range at the fom psi-lab[END_REF][START_REF] Holtkamp | The status of the iter design[END_REF]1]. Magnum-PSI uses a cascaded arc as plasma source which is similar in basic design to the source used in this article [START_REF] De Groot | Magnum-psi, a new linear plasma generator for plasma-surface interaction studies in iter relevant conditions[END_REF][START_REF] Kroesen | Description of a flowing cascade arc plasma[END_REF][START_REF] Van De Sanden | A combined thomson-rayleigh scattering diagnostic using an intensified photodiode array[END_REF]. Moreover, this source design is used in both research and industry for the removal, deposition or modification of functional materials such as amorphous silicon nitride (a-SiN x :H) and hydrogenated amorphous carbon (a-C:H) [START_REF] Gielen | Optical and mechanical properties of plasmabeam-deposited amorphous hydrogenated carbon[END_REF][START_REF] Robertson | Diamond-like amorphous carbon[END_REF][START_REF] Benedikt | Plasma chemistry during the deposition of a-c:h films and its influence on film properties[END_REF][START_REF] Yu | Chemical kinetics in carbon depositing d.c.-arc jet cvd reactors[END_REF][START_REF] Hoex | SHORT COMMUNICATION Industrial highrate ( 5 nm/s) deposited silicon nitride yielding high-quality bulk and surface passivation under optimum anti-reflection coating conditions[END_REF][START_REF] Weber | Spectroscopic ellipsometry on hydrocarbon erosion by plasma jets[END_REF].

The expanding thermal plasma generated by a cascaded arc has been extensively investigated in the past (see section 2). Typical for this type of plasma are the high fluxes (up to 10 24 m -2 s -1 , depending on the plasma source and reactor configuration) and low electron and ion kinetic temperature (∼ 1 eV) near the arc's nozzle. These temperatures decrease to about 0.1 eV near the target. Furthermore, applying a bias voltage to the target increases the kinetic energy of the incident ions to iter relevant values [START_REF] Von | Growth and erosion of hydrocarbon films investigated by in situ ellipsometry[END_REF][START_REF] Roth | Flux dependence of carbon chemical erosion by deuterium ions[END_REF][START_REF] Kleyn | Plasma-surface interaction in iter[END_REF][START_REF] Kudlacek | Accurate control of ion bombardment in remote plasmas using pulse-shaped biasing[END_REF]. These plasma properties -i.e. high ion fluxes and low plasma temperatures -fall in the intersection between typical high-temperature experimental fusion research and low-temperature industrial plasma processing. Based on our experience in low-temperature plasma processing, we can provide insight into the plasma-surface interaction between (relatively) high flux, low-temperature plasmas and a-C:H thin film samples as a model for carbon erosion. Surface processes that occur during a-C:H erosion under such plasma conditions are largely unexplored and will be treated extensively in a forthcoming paper. Those results will indicate ion-assisted chemical erosion as the dominant erosion process, even at ion kinetic energies around 1 -2 eV [START_REF] Annen | Chemical erosion of amorphous hydrogenated boron films[END_REF]. This article deals with the plasma chemistry during a-C:H and graphite erosion, which is dominated by charge transfer between ions and molecular species [START_REF] Beulens | Atomic and molecular emission spectroscopy on an expanding argon/methane plasma[END_REF][START_REF] Gielen | Plasma beam deposited amorphous hydrogenated carbon: Improved film quality at higher growth rate[END_REF][START_REF] De Graaf | Argon ion-induced dissociation of acetylene in an expanding ar/c2h2 plasma[END_REF][START_REF] R Engeln | Detection of ch in an expanding argon/acetylene plasma using cavity ring down absorption spectroscopy[END_REF].

Charge transfer (ct), followed by dissociative recombination with an electron (dr), leads to a reduction in ion density as well as molecular fragmentation. The ion density and flux towards the surface are important factors in plasma processing. Similar, the resulting molecular fragments can either etch the target or (re-)deposit on it. Furthermore, gas recirculation cells inside a linear plasma reactor play an important role in the chemistry of the entire system [START_REF] Meulenbroeks | Wall association and recirculation in expanding thermal arc plasmas[END_REF][START_REF] Meulenbroeks | Coherent anti-stokes raman scattering performed on expanding thermal arc plasmas[END_REF][START_REF] Mazouffre | Transport of ground-state hydrogen atoms in a plasma expansion[END_REF][START_REF] Engeln | Flow dynamics and invasion by background gas of a supersonically expanding thermal plasma[END_REF][START_REF] Gabriel | Two-dimensional flow characteristic of a hot expanding plasma[END_REF]. These recirculation cells lead to a complete mixture of the background gas, while diffusion back and forth between the plasma expansion and background gas connects both regions. The flow dynamics of both ions and molecular species plays a role in where and when molecular fragments are produced and can therefore affect the local plasma chemistry as well as the surface processes. A solid understanding of the gas dynamics and plasma chemistry in a linear plasma reactor is thus important for correctly interpreting experimental results obtained with such a system. Therefore, in addition to the plasma chemistry, this paper will also investigate the flow dynamics. In particular, the effect of a substrate as well as the retraction or extension of a shutter in front of the a-C:H sample on the flow dynamics and the local plasma chemistry are investigated.

As can be clearly seen in the photographic images by van Helden et al., a substrate deflects an expanding thermal plasma [START_REF] Van Helden | Molecule formation in n and o containing plasmas[END_REF]. The presence of a substrate in a plasma expansion may also lead to changes in the recirculation cells and thus affect where the diffusion of the background gas into the plasma occurs. The influence of a substrate is therefore investigated via spatially resolved measurements of the ion density in an Ar/H 2 plasma, both with and without a substrate. The contours of the expanding thermal plasma and its deflection on the substrate will be visualized with these measurements.

The presence of a substrate, moreover, increases the ion density as will be shown. Next, these same plasma conditions are used for the erosion of both a-C:H and graphite targets.

It will be shown that ct and dr between the hydrocarbon etch products of both targets and Ar + ions leads to the formation of ro-vibrationally excited CH radicals. The optical emission of these CH radicals consequently acts as a tracer for both the argon ions and the hydrocarbon etch products [START_REF] Westerhout | Ch spectroscopy for carbon chemical erosion analysis in high density low temperature hydrogen plasma[END_REF]. Spatially resolved measurements of the CH emission are used to determine the hydrocarbon redistribution near the vicinity of the target. Furthermore the rotational and vibrational temperatures of the CH radical are strongly affected by the reactor conditions which, in turn, are substantially influenced by the retraction or extension of a shutter in front of the a-C:H sample. The changes caused by both the substrate and shutter in either the ion density or the ro-vibrational CH temperature suggest a reorganisation of the recirculation pattern. Measurements related to the plasma chemistry are thus instrumental in investigating the gas flow and recirculation pattern in a linear plasma reactor. These issues, i.e. pertubations of the flow dynamics and the local plasma chemistry, should be considered in all linear plasma reactors prior to investigating plasma-surface interactions.

Flow dynamics of an expanding thermal plasma

Both the flow dynamics of and the plasma chemistry in an expanding thermal plasma have been extensively investigated in the past and are briefly described in this section.

Gas injected under high pressure into a cascaded arc is ionized by a dc current.

With a typical ionization degree of around 10%, the generated plasma consists of neutral species as well as ions and electrons. This plasma expands supersonically into the low pressure reactor until it encounters the background gas, at which point a barrel shock is created. After the shock, the plasma continues subsonically towards the substrate or reactor walls. The shock position z s (in mm) on the plasma axis can be estimated from:

z s = 18 Φ P bg 4 √ MT , (1)
with the gas flow Φ in sccs (1 sccs = 2.5 × 10 19 s -1 ), the background pressure P bg in Pa, the mass M in amu and the temperature of the plasma source T in eV [START_REF] Vankan | Inflow and shock formation in supersonic, rarefied plasma expansions[END_REF]. The plasma expansion is furthermore radially symmetric and quasi-neutral (n e = n ion ), but not in local thermal equilibrium [START_REF] Kroesen | Description of a flowing cascade arc plasma[END_REF].

Four regions have to be considered when discussing the thermal properties of the plasma. First: the electron, ion and neutral gas kinetic temperatures are in equilibrium within the arc itself, which is the only active region in the entire reactor. In other words, the arc produces a thermal plasma with an electron temperature of about 1 eV near the nozzle. Second: due to the low ionization degree, the supersonic part of the expansion behaves similar to that of a hot gas expansion [START_REF] Engeln | Flow dynamics and invasion by background gas of a supersonically expanding thermal plasma[END_REF]. The ions cool down adiabatically, thereby converting thermal energy into forward directed kinetic energy [START_REF] Kroesen | Description of a flowing cascade arc plasma[END_REF][START_REF] Van De Sanden | Recombination of argon in an expanding plasma-jet[END_REF][START_REF] Otorbaev | Spectroscopic measurement of atomic-hydrogen level populations and hydrogen dissociation degree in expanding cascaded arc plasmas[END_REF]. The electron kinetic temperature in the supersonic part furthermore decouples from the ion and neutral gas kinetic temperature since the energy exchange between heavy particles occurs more efficiently than between electrons and heavy particles [START_REF] Kroesen | Description of a flowing cascade arc plasma[END_REF]. Third: the temperature increases again across the shock due to the back conversion of forward directed kinetic energy into random thermal energy [START_REF] Mazouffre | Transport of ground-state hydrogen atoms in a plasma expansion[END_REF]. Fourth: particle transport in the subsonic part of the plasma expansion, i.e. the region of interest in this paper, is controlled by diffusion [START_REF] Mazouffre | Transport of ground-state hydrogen atoms in a plasma expansion[END_REF]. This leads to heat loss at the wall and a subsequent temperature decrease. In this part of the plasma expansion, the electron, ion and neutral gas kinetic temperatures have thermalized and are on the order of 0.1 eV [START_REF] Van De Sanden | Recombination of argon in an expanding plasma-jet[END_REF][START_REF] Meulenbroeks | Fabry-p[e-acute]rot line shape analysis on an expanding cascaded arc plasma in argon[END_REF].

The gas residence time in the reactor can be calculated from the gas flow Φ (in sccs), the pressure P (in Pa) and reactor volume V (in m 3 ) with τ = 10×P V Φ and is on the order of a second during the experiments discussed in this work. Within the expansion itself, however, the ions and electrons have a residence time on the order of 0.1 ms [START_REF] Meulenbroeks | Wall association and recirculation in expanding thermal arc plasmas[END_REF].

The much higher residence time of the background gas causes the background gas to recirculate and interact multiple times with the reactor wall. The resulting recirculation cells, wedged between the plasma expansion and the reactor wall, are also responsible for a good mixture of the background gas (figure 1). This gas can moreover diffuse into the plasma over the entire length of the expansion and interact with the electrons and ions emanating from the arc [START_REF] Gabriel | Two-dimensional flow characteristic of a hot expanding plasma[END_REF]. In the supersonic part of the expansion, background species can enter through the barrel shock if their partial pressure inside the expansion is lower than in the background [START_REF] Engeln | Flow dynamics and invasion by background gas of a supersonically expanding thermal plasma[END_REF][START_REF] Gabriel | Two-dimensional flow characteristic of a hot expanding plasma[END_REF]. This is particularly the case in the region right before the shock front, where the plasma is overexpanded [START_REF] Mazouffre | Transport of ground-state hydrogen atoms in a plasma expansion[END_REF]. The total pressure in this so-called zone of silence is lower than the overall background pressure.

Experimental setup

The cascaded arc, as used in this paper, consists of a stack of four water-cooled copper plates with a central bore of 4 mm in diameter and has a total length of 24 mm (figure 1) [START_REF] Kroesen | Description of a flowing cascade arc plasma[END_REF][START_REF] Van De Sanden | A combined thomson-rayleigh scattering diagnostic using an intensified photodiode array[END_REF]. The arc is operated on a 60 A dc current, a gas flow of 50 sccs argon and a pressure of about 600 mbar. A constant background pressure of 100 Pa is maintained inside the reactor itself by regulating the opening of the pump valve, i.e. the effective pumping speed. Molecular hydrogen (H 2 ) with a gasflow of 1 sccs is moreover injected directly in the background of the reactor via a second gas inlet. The shock position under these conditions is located ∼ 32 mm from the nozzle, irrespective of the small H 2

inflow. An electrically floating substrate is furthermore positioned at ∼ 320 mm from the nozzle.

This arc configuration is used on two different reactors. Reactor one has an arc and substrate which can move independently with respect to a double Langmuir probe and each other. This double probe measures the ion density with a spatial resolution of 5 mm in both radial and axial direction. Length and diameter of the tungsten wires are 3.85 ± 0.05 mm and 0.85 ± 0.05 mm, respectively. Spacing between the wires is likewise 0.85 ± 0.05 mm. A Keithley 2400 source meter provides a voltage sweep from -5 V to 5 V in steps of 0.025 V and measures the current. The resulting I-V characteristic, averaged over 5 voltage sweeps, is analysed according to the theory by Peterson and Albot [START_REF] Peterson | Collisionless electrostatic single-probe and double-probe measurements[END_REF].

Reactor two has an arc and substrate with a fixed position ( For more details on the deposition process itself, see [START_REF] Weber | B-spline parametrization of the dielectric function applied to spectroscopic ellipsometry on amorphous carbon[END_REF] and references therein. The second type of target is a commercially available fine-grain graphite block (fgg; R6650, sgl Carbon group) with a diameter of 30 mm. This is 45 mm smaller than the target holder itself, i.e. the substrate.

The erosion process of the carbon targets is monitored by residual gas analysis (rga; Pfeiffer Vacuum QMS 200) and by optical emission spectroscopy (oes) [START_REF] Dingemans | Probing the phase composition of silicon films in situ by etch product detection[END_REF]. Light from the plasma is collected during oes measurements by a lens (focal length = 5.7 ± 0.2 cm) and focused onto an optical fiber and transported to a commercially available spectrometer (AvaSpec-2048-4-DT, Avantes), resulting in a line-of-sight measurement.

The wavelength resolution of the overview channel (150 -1300 nm) is 1.3 nm and 0.3 nm for the high resolution channel (350 -610 nm). The time resolution is 1 and 2.5 s during the plasma etching of a-C:H and FGG, respectively. Of all the different carbonbased molecules present in our reactor, only the emission of CH and C 2 falls within the wavelength range of our spectrometer as can be seen in figure 2. Moreover, the rotational (T rot ) and vibrational (T vib ) temperature of the electronic CH(A-X) transition can be determined by means of the spectrum simulation program lifbase [START_REF] Luque | Lifbase: Database and spectral simulation program (version 1.9)[END_REF][START_REF] Luque | Electronic transition moment and rotational transition probabilities in ch .1. a (2)delta-x(2)pi system[END_REF]. Figure 3, as an illustration, shows a measured CH spectrum together with the corresponding fit. Rather than fitting all the spectra manually, the method as explained in appendix Appendix B is used to determine the rotational and vibrational temperature of each spectrum.

Experimental results

The experimental results are divided into four sections. The first section discusses the ion composition and effective ion mass of the plasma. Section (4.2) deals with the influence of a substrate on the ion density in an Ar/H 2 plasma. The (re-

)distribution and dissociation of hydrocarbon etch products is discussed in section 4.3.

A connection between the ro-vibrational temperatures of the CH radical and the gas kinetic temperature will be show in the last section, i.e. 4.4.

Ion composition of the plasma

The ion composition and plasma chemistry in pure argon and argon/hydrogen expanding thermal plasmas have been studied in the past by our group and is therefore not extensively treated here [START_REF] Kroesen | Description of a flowing cascade arc plasma[END_REF][START_REF] Van De Sanden | A combined thomson-rayleigh scattering diagnostic using an intensified photodiode array[END_REF][START_REF] De Graaf | Anomalous fast recombination in hydrogen plasmas involving rovibrational excitation[END_REF][START_REF] Otorbaev | Spectroscopic measurement of atomic-hydrogen level populations and hydrogen dissociation degree in expanding cascaded arc plasmas[END_REF][START_REF] Meulenbroeks | The argon-hydrogen expanding plasmamodel and experiments[END_REF][START_REF] Vankan | High rotational excitation of molecular hydrogen in plasmas[END_REF]. These studies showed that ionization or dissociation of neutral species via electron impact only occurs in the arc, where the electron temperature and density are high enough. Nevertheless, charge transfer and dissociative recombination within either the arc or the reactor likewise leads to the formation of new species. Reaction rates for charge transfer are overall lower than for dissociative recombination by 2 or 3 orders of magnitude. Charge transfer is thus the time-limiting factor in this process. H 2 dissociation and ion formation via ct and dr in Ar/H 2 plasma is explained in appendix Appendix A.

The effective ion mass is a required input parameter in the analysis of the double Langmuir probe data when using the Peterson and Albot theory. This is 40 amu for Ar + in a pure argon plasma. The ion composition in an Ar/H 2 plasma consists of Ar + , ArH + and H + 3 and depends strongly on the gas mixture as discussed in appendix Appendix A.

The exact ion composition and thus the effective ion mass for the plasma conditions in this work remains however unclear. An ion mass of 40 amu is therefore used as well for the Ar/H 2 measurements in section 4.2, although this approach yields on overestimation of the ion density by up to a factor of four. Immediately noticeable is that the highest densities (> 3.8 × 10 18 m -3 ) extend further into the reactor, towards the substrate. The density at 40 mm is about 50% higher when the substrate interacts with the plasma expansion. The last two to three data points in front of the substrate, on the other hand, show a relatively larger decline in density. Notice furthermore the appearance of indentations and protuberances on the plasma boundary and the deflection of the plasma on the substrate. The influence of a substrate on the ion density is further discussed in section 5. deposition precursors leads to the formation of excited CH radicals [START_REF] R Engeln | Detection of ch in an expanding argon/acetylene plasma using cavity ring down absorption spectroscopy[END_REF]. Figure 5 shows the relative intensity of the Q(0,0) band head of CH(A-X) transition at 431 nm with a spatial resolution of 5 mm. The scale on the axis is limited by the size of the viewport and, therefore, smaller than in figure 4. As can be seen in figure 5, CH radicals are likewise found throughout the reactor during carbon erosion (fine-grain graphite or fgg in this case). With a thermal velocity of ∼ 1 km s -1 and a lifetime of 500 to 700 ns for the first excited state, the CH radical can travel up to 0.77 mm before it decays [START_REF] Luque | Electronic transition moment and rotational transition probabilities in ch .1. a (2)delta-x(2)pi system[END_REF]. This short travel distance excludes the carbon surface as the origin of these excited radicals. Electron ionisation of existing CH radicals in the gas phase is likewise excluded due to the low electron temperature of the plasma. CH emission thus occurs in the immediate vicinity of its production, i.e. through ct and dr reactions between Ar + and hydrocarbons such as CH 4 or C 2 H 2 .

Influence of a substrate on the plasma expansion

Spatial and time resolved CH emission during carbon erosion

A
A mean free path (mfp) for these hydrocarbon etch products is estimated from their thermal velocity, the reaction rate for ct and the ion density. The thermal velocity is around 1 km s -1 , while the reaction rate is on the order of 10 -16 to 10 -15 m 3 s -1 for C 1,2 H y [START_REF] De | Deposition of CNH Materials: Plasma and Film Characterization[END_REF][START_REF] Benedikt | Detailed tims study of ar/c2h2 expanding thermal plasma: Identification of a-c:h film growth precursors[END_REF]. Acetylene, for instance, would have a mfp on the order of 10-30 cm in a pure argon plasma with an ion density of ∼ 2 × 10 19 m -3 (figure 4.a) [START_REF] Gielen | Plasma beam deposited amorphous hydrogenated carbon: Improved film quality at higher growth rate[END_REF][START_REF] Benedikt | Detailed tims study of ar/c2h2 expanding thermal plasma: Identification of a-c:h film growth precursors[END_REF]. (ii) The e-folding length of the measured intensity on the z-axis is 6.0±0.5 mm, which is significantly smaller than the previously calculated mean free path of up to 100 cm for methane. Similar measurements conducted by Westerhout et al. in a pure hydrogen plasma showed a good correspondence between the e-folding length in the z-direction of the CH emission and the mean free path of methane, injected via the substrate [START_REF] Westerhout | Ch spectroscopy for carbon chemical erosion analysis in high density low temperature hydrogen plasma[END_REF]. The neutral density in our case is however one order of magnitude higher, which increases the number of neutral -neutral collisions. This reduces the effective travel distance of the etch products prior to ct and dr and is thus expected to yield a smaller e-folding length.

(iii) Emission levels are lower at the edges of the fgg target, i.e. at a radial position of about ±20 mm, compared to the immediate surroundings. This indicates that the region around the sides of the 4 mm thick target is poor in either Ar + ions or hydrocarbons.

(iv) The pump duct located at the bottom of the back flange, i.e. below the target, is responsible for the asymmetry in the measured intensity at larger radial positions (> 40 mm).

(v) The CH emission accentuates the central axis of the plasma expansion for z > 10 mm's. It also shows the deflection of the plasma expansion by the substrate.

(vi) The spectrum at each location was measured several times during the measurement, which lasted over an hour. Nevertheless, the variation in intensity is less than 3%.

The (re-)distribution of hydrocarbons in the ground state, which can not be detected via optical emission spectroscopy, is furthermore determined via numerical simulations of the gas flow in the reactor. The DS2V code developed by G.A. Bird is a direct simulation Monte Carlo (dsmc) method and has been previously used to describe the plasma expansion [START_REF] Selezneva | Stationary supersonic plasma expansion: continuum fluid mechanics versus direct simulation monte carlo method[END_REF][START_REF] Gabriel | Two-dimensional flow characteristic of a hot expanding plasma[END_REF][START_REF] Abbate | Multi-scale Modelling of the Two-Dimensional Flow Dynamics in a Stationary Supersonic Hot Gas Expansion[END_REF]. Our simulation starts from a fully developed expansion of a pure argon gas. After a stable gas flow has been calculated, a secondary gas inlet of methane-like particles is added to the model at the position of the substrate to simulate the erosion of a carbon target. The injected flow ratio of argon to methane is a 100:1. The model contains neither charged particles, nor any gas phase chemistry. The results presented in figure 6 show the gas expansion and its deflection on the substrate.

Cooling of the gas along the main axis can likewise be seen. In contrast with the CH emission, moreover, the injected methane-like particles are primarily found in front of the substrate and in the background of the reactor. The distribution in the CH emission, as measured in figure 5, is thus due to the convolution of the Ar + ion density and the hydrocarbon density throughout the reactor.

A thick fgg target, as used so far, enables steady state measurements. The target type is now switched to a-C:H thin films for the study of time dependent effects in the gas flow. Optical emission from the plasma is measured a few mm's in front of an a-C:H sample with a time resolution of 1 s. Note that the focus of the optical system in these measurements is located in between sample and shutter (as can be seen in figure 9 later on). This implies that little to none emission of any wavelength is recorded by the spectrometer when the plasma is blocked by the shutter. Figure 7 shows the time evolution of the CH peak at 431 nm. When the shutter is retracted at 0 s, the measured intensity increases sharply. Afterwards the intensity increases steadily for about 220 s due to an increased etch rate caused by plasma heating of the substrate. Maximum intensity in the emission is reached a few seconds before the sample is completely eroded as indicated by spectroscopic ellipsometry. Or in other words, CH emission starts to decrease as the hydrocarbon source (i.e. the a-C:H surface) is depleted. Between ∼ 250 s and ∼ 470 s, the measured intensity remains high due to a continuous re-deposition and re-erosion and due to the etching of carbon impurities on the reactor walls. Extending the shutter at ∼ 470 s blocks the plasma expansion again, which strongly reduces the overall light emission at the focal point of the optical measurement system. This verifies that CH emission is not measured when Ar + ions and electrons are unavailable for ct and dr at the focal point.

Rotational and vibrational temperature of CH

The method described in section Appendix B will now be used to determine the rotational and vibrational temperature of the CH radical for the previously discussed spatial and time resolved measurements.

Spatially resolved Engeln et al. measured the density of CH in the ground state

during deposition conditions, thereby assuming thermal equilibrium between CH and the background gas [START_REF] R Engeln | Detection of ch in an expanding argon/acetylene plasma using cavity ring down absorption spectroscopy[END_REF]. The rotational temperature in particular was assumed to be a measure of the gas kinetic temperature (T gas ) [START_REF] R Engeln | Detection of ch in an expanding argon/acetylene plasma using cavity ring down absorption spectroscopy[END_REF]. Ro-vibrational temperatures of the first electronically excited state CH(A-X), as shown in figure 8, are on the other hand higher than the gas kinetic temperature of 1000 -2000 K. The highest temperatures are measured on the central axis of the plasma expansion and nearest to the cascaded arc. Both T rot and T vib decrease in axial direction towards the substrate as well as in radical direction. A similar trend for the gas temperature, seen in the bottom part of figure 6, indicates a connection between the electronically excited state and T gas as well. The lifetime of the excited state (section 4.3) however provides insufficient time to equilibrate with the background gas. A memory effect is therefore assumed between the ro-vibrational temperature of the electronically excited CH and the gas kinetic temperature by proxy of the CH parent molecule.

Time resolved

Figure 9, which corresponds with figure 7, shows the time variation in the rotational and vibrational temperature. The intensity measured after 250 s is too low to accurately determine these two temperatures. A slow increase from ∼ 4700 K to ∼ 4800 K can be observed in figure 9.a. T rot , on the other hand, decreases exponentially from ∼ 3200 K to ∼ 2200 K. This decrease is explained in detail in section 5. Two spectra -averaged over 10 measurement points and normalized to the Q(0,0) band head -obtained near the beginning and end of the a-C:H erosion experiment are shown in figure 10. A variation in peak intensities relative to the Q(0,0) band head provides visual confirmation of a change in the ro-vibrational CH(A-X) temperature (see eq. 2.1a and 2.1b in App. Appendix B). The larger variation in T rot between beginning and end of the measurement suggests that the rotational energy levels are more sensitive to the gas temperature than the vibrational temperature. This can be expected in a low-temperature plasma since the rotational energy spacings (∼ 0.01 eV) are smaller than the vibrational energy spacings (∼ 0.2 eV) [START_REF] Lieberman | Principles of Plasma Discharges and Materials Processing[END_REF].

Discussion and conclusions

Placing a substrate inside the expanding thermal plasma has a clear effect on the ion density and the shape of the plasma expansion as shown in section 4.2. The lower ion density in front of the substrate is attributed to the formation of a stagnation zone with a thickness on the order of 10 mm in axial direction. An increased neutral particle (i.e. H 2 ) density in this stagnation zone increases the number of ion-neutral collisions via ct and thus also the ion losses. This effect can moreover be seen in the CH emission, whereby the excited CH radicals themselves are formed via ct and dr between Ar + and hydrocarbon etch products. As stated in section 4.3, the intensity of the CH emission is due to the convolution of the Ar + ion density and the hydrocarbon density throughout the reactor. An increased pressure in the stagnation zone augments the local hydrocarbon density, which is already high due to the vicinity of the carbon target. The combination of a substrate and a deflected plasma expansion furthermore leads to a restructuring of the recirculation cells in the background. Such a restructuring is moreover considered to be responsible for the reduced decline in ion density further upstream from the substrate. ct reactions outside of the stagnation zone require a molecular gas inflow from the background (or alternatively from the arc, which is not the case here). Previous work in our group showed a bimodal component in the velocity distribution of argon for a pure argon plasma [START_REF] Gabriel | Two-dimensional flow characteristic of a hot expanding plasma[END_REF]. A hot, fast component originating from the cascaded arc and a slow, cool component from the inflowing background gas.

Impeding this gas inflow via a restructured circulatory system in the background thus reduces the ion losses as was observed.

The vibrational and rotational temperature of CH in section 4.4 likewise provide information about the plasma expansion and indirectly about the recirculation cells.

The steady-state measurements, first of all, visualises the plasma cooling in both radial and axial direction, thereby relating the rotational and vibrational temperature of CH to the gas kinetic temperature in the expansion via the CH parent molecule. The plasma expansion is furthermore deflected by the shutter towards the walls, thereby displacing the recirculation cells (figure 11). The deflected plasma will subsequently heat up both the wall and the background gas. When the shutter is retracted to start the etch process, the walls and the background gas in the reformed recirculation cells no longer receive an energy influx from the plasma and start to gradually cool down.

As the background cools down over time, the inflowing gas will likewise have a lower kinetic energy. This temporal variation in the gas kinetic temperature is reflected in the time-dependent rotational temperature of CH, as observed in section 4.4. Or in other words, the rotational temperature of the CH radical acts as a thermometer for changes in the background environment.

In conclusion, the distribution of electronically excited CH radicals, locally produced via charge transfer and dissociative recombination reactions, is due to the convolution of the Ar + ion density and the hydrocarbon density throughout the reactor.

Moreover, the rotational temperature of electronically excited CH is related to the gas kinetic temperature via their parent molecules. Variations in the local ion density or gas temperature therefore affect the production of excited species which in turn can have an impact on the local plasma chemistry and surface processes. Both the placement of a substrate and the movement of a shutter inside the plasma expansion are two external influences that cause such variations via a reorganisation of the recirculation cells. The ion density is likewise affected via the formation of a stagnation zone in front of the substrate. Plasma etching of carbon, in particular hydrogenated amorphous carbon, is thus well suited to investigate the local plasma chemistry and identify (externally caused) pertubations in the gas flow of a linear plasma reactor.

Likewise, the gas flow circulatory system in Magnum-PSI -and iter, later onshould be investigated to account for its effects on the erosion process. Work in that direction has already been started by van Eck et al. and Westerhout et al. [START_REF] Westerhout | Ch spectroscopy for carbon chemical erosion analysis in high density low temperature hydrogen plasma[END_REF][START_REF] Van Eck | A differentially pumped argon plasma in the linear plasma generator magnumpsi: gas flow and dynamics of the ionized fraction[END_REF].

Furthermore, the presence of magnetic fields in both Magnum-PSI and iter will affect the flow of the plasma. In particular, movement perpendicular to the magnetic field lines will be restricted. A continuation of this paper, therefore, would be an investigation of composition in an Ar/H 2 plasma is therefore discussed in this appendix.

Charge transfer between Ar + ions and H 2 followed by the associated dissociative recombination reactions leads to the production of atomic H at the expense of the Ar + ions. This however occurs via ArH + and H + 2 as intermediate ions as can be seen in the reactions:

Ar + + H 2 → ArH + + H (1.1a) → Ar + H + 2 , (1.1b)
Reactions (1.1a) and (1.1b) have a reaction rate of 1.2 × 10 -15 m 3 s -1 and 1.7 × 10 -16 m 3 s -1 , respectively [START_REF] Otorbaev | Spectroscopic measurement of atomic-hydrogen level populations and hydrogen dissociation degree in expanding cascaded arc plasmas[END_REF]. The first reaction is dominant as reaction (1.1b) is of little importance below 0.5 eV [START_REF] Meulenbroeks | Argon-hydrogen plasma-jet investigated by active and passive spectroscopic means[END_REF]. A third possible ct reaction with a rate of ∼ 10 -15 m 3 s -1 is given by [START_REF] De Graaf | Anomalous fast recombination in hydrogen plasmas involving rovibrational excitation[END_REF][START_REF] Vankan | High rotational excitation of molecular hydrogen in plasmas[END_REF]:

H + 2 + H 2 → H + 3 + H. (1.2)
The different dr reactions, not shown here, all have a reaction rate around ∼ 10 -13 m 3 s -1 . Reaction (1.2)) thus starts to play a role when the n H 2 over n e -ratio exceeds 100, as can be seen from the reaction rates.

The ion composition of the plasma, which in turn determines the effective ion mass, is thus to a large degree dependent on the n H 2 over n e -ratio. Even so, the transition between a purely Ar + and a purely H + 3 ion composition should be smooth.

IV-characterics are therefore measured as function of the H 2 gas flow admixed to an argon plasma and analysed for a range of ion masses: 3 and 38-41 amu (figure 12).

Note that an ion mass of 40 amu is always used for 0% H 2 , while a mass of 41 amu represents an ion composition of 100% ArH + . These measurements were taken in the center of the reactor at a distance of 31.5 cm from the plasma source, without the presence of a substrate. First of all, the difference in ion density is about 3 to 4% when analysed with a mass of 38 to 41 amu. Analysis of the data for a mass of 41 amu yields a density four times higher than for a mass of 3 amu. A factor of four difference would thus constitute the worst case scenario, not taking into account any other measurement errors. The graph furthermore shows the strongest decline in ion density between 0 and 2%, regardless of ion mass. Extrapolating this initial decline (for 40 amu) would yield a 100-fold decrease in ion density for a gas mixture with ∼ 2.2% H 2 . The measured ion density for gas mixtures with more than 2% H 2 however remains higher than expected from a linear consumption of Ar + ions. This is attributed to H + 3 formation, which counteracts the loss of Ar + in the total ion density. Previous work in our group also showed that more than 3% of H 2 in the gas flow leads to a drastic change in the ion composition or in the behaviour of the arc [START_REF] Meulenbroeks | The argon-hydrogen expanding plasmamodel and experiments[END_REF][START_REF] Kessels | Formation of cationic silicon clusters in a remote silane plasma and their contribution to hydrogenated amorphous silicon film growth[END_REF]. A gas mixture of ∼ 2% of H 2 , as used in this work, falls within the transition from an Ar + dominated to an H + 3 dominated plasma.

Appendix B. Fitting CH(A-X) spectra

The spectrum simulation program Lifbase can determine the rotational and vibrational temperature of the electronic CH(A-X) transition. Manually fitting each spectrum is however not always feasible when large data sets are involved. Beulens et al. observed that T vib , fortunately, depends heavily on the ratio of the Q(0, 0) and Q(2, 2) band heads, more so than T rot [START_REF] Beulens | Atomic and molecular emission spectroscopy on an expanding argon/methane plasma[END_REF]. This allows for a separation between the rotational and vibrational temperatures based on the relative peak intensity. Therefore, the following empirical approach can be used. First, lifbase is used to simulate a set of CH(A-X) spectra for a wide range of rotational and vibrational temperatures. Two proportionality factors are then determined from this set:

P vib = Q(2, 2) Q(0, 0) (2.1a) P rot = 431 nm 415 nm R(i, f) Q(0, 0) , (2.1b)
with i and f the initial and final vibrational state, respectively. The Q(0, 0) band head is included in the sum of (2.1b) as well. The difference in rotational quantum number (∆J) is 1 for the P-branch, 0 for the Q-branch and -1 for the R-branch. Second, two analytic expressions are obtained for these proportionality factors as function of the simulated temperature (figure 13):

P vib = a + b × ln(T vib + c) ( 2 . 2 a) P rot = d × T rot + e, (2.2b)
with the inverse relations given by:

T vib = a × e P vib b + e (2.3a)
T rot = d × P rot + e. (2.3b)
Table 1 lists the fit parameters a through e for both sets of equations. The third and last step is to determine the ratios P vib and P rot for each measured spectrum. Both temperatures are then calculated from these measured ratios. . The initial decline in ion density extrapolates to a > 100-fold decrease at a gas flow of ∼ 2.2% when assuming a linear consumption of Ar + ions. The higher than expected ion density above this gas flow of ∼ 2.2% is attributed to H + 3 formation, which counteracts the loss of Ar + in the total ion density.

  Fig 11). The substrate temperature is set to a starting value of 250 • C, which increases further due to plasma heating. Two types of carbon-based targets are exposed in this reactor to an Ar plasma, admixed with 1 sccs of H 2 . Hydrogenated amorphous carbon samples (a-C:H) are the first type of target. These samples are pre-deposited in-house by means of plasmaenhanced chemical vapour deposition[START_REF] Gielen | Quality improvement of plasma-beam-deposited amorphous hydrogenated carbon with higher growth rate[END_REF][START_REF] Singh | A hard graphite-like hydrogenated amorphous carbon grown at high deposition rate[END_REF]. During deposition, the arc current is 75 A with an argon gas flow of 100 sccs. The substrate temperature and background pressure during sample preparation are 250 • C and 31 Pa, respectively. The deposited samples are about 1 µm thick. After deposition, the samples are stored in a desiccator.

Figure 4

 4 Figure4shows the spatial distribution of the ion density for three different plasma

Figure 4 .

 4 Figure 4.c shows the influence of a substrate on the ion density in an Ar/H 2 plasma.

Figure 4 .

 4 Figure 4.c in turn yields a much lower ion density of 1 -5 ×10 18 m -3 for an Ar/H 2

  the gas flow dynamics in a magnetized plasma. Possible research questions include: (a) how and to which extent (external) perturbations to the magnetic field -in analogue of the displacement of a shutter in this paper -affect the gas flow dynamics and (b)what are the consequences thereof on the (local) plasma chemistry and ro-vibrational CH temperatures. In our view, such a study can and should be carried out at the Magnum-psi and similar facilities to better characterize the reactor in terms of the gas flow dynamics and the influence thereof on erosion studies.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: (Colour online) Schematic cross-section of the reactor with movable plasma source and substrate. Recirculation cells are responsible for a complete mixture of the background gas.

Figure 4 :Figure 5 :

 45 Figure 4: (Colour online) Cross-section of the ion density (in m -3 ) for a) a pure argon plasma, b) an argon plasma admixed with 2% H 2 and the same Ar/H 2 plasma c) with a substrate at floating potential. Note the difference in scale between frame a and frames b and c.

Figure 6 :

 6 Figure 6: (Colour online) Numerical simulation of the plasma expansion. Top image shows the distribution in the reactor of a methane-like gas, injected at the substrate. Bottom image shows the temperature in the reactor. The expansion and its deflection on the substrate are clearly visible.

Figure 7 :

 7 Figure 7: Evolution of the CH(A-X) emission a few mm's in front of an a-C:H sample. The time resolution is 1 s. The gray bar indicates the uncertainty in when the sample is completely eroded according to spectroscopic ellipsometry. The elevated emission after 250 s is due to wall impurities.

Figure 8 :

 8 Figure 8: (Colour online) Spatially resolved a) vibrational and b) rotational temperature of CH(A-X) during plasma erosion of a fgg target. See also figure 5.

Figure 9 :Figure 10 :

 910 Figure 9: Rotational and vibrational temperature of CH(A-X) during plasma erosion of an a-C:H sample, corresponding to figure 7. The measured spectra at points a and b (red circles) are shown in figure 10.

Figure 11 :Figure 12 :

 1112 Figure11: Schematic cross-section of the reactor with fixed arc and substrate. The plasma is deflected by the shutter towards the wall, effectively dividing the reactor into two. The focus of the optical system falls in between the shutter and the substrate.

Figure 13 :

 13 Figure 13: (Colour online) Proportionality factors for simulated rotational and vibrational temperatures of the CH(A-X) emission band. The fit functions are given by equations (2.2a) and (2.2b).

  similar Ar/H 2 plasma as shown in figure 4.b and figure 4.c is now used for the erosion of both graphite and a-C:H targets. Stable hydrocarbon species detected by rga during plasma exposure of these two different targets are mainly CH 4 and C 2 H

y (y = 2, 4). Previous work in our group has shown that ct and dr with hydrocarbon

Table 1 :

 1 Fit parameters for the equations (2.2a) to (2.3b).

		(2.2a)	( 2 . 3 a)
	a	-5.28 ± 0.57	5729 ± 640
	b	0.600 ± 0.06	0.537 ± 0.045
	c	4959 ± 816	-4024 ± 664
		(2.2b)	( 2 . 3 b)
	d	(2.10 ± 0.03) × 10 -4	4737 ± 63
	e	2.04 ± 0.02	-9638 ± 181

Acknowledgments

We greatly appreciate the skillful technical assistance of M.J.F. van de Sande, J.J.A. Zeebregts and H.M.M. de Jong. This work is part of the research program of the Dutch Foundation for Fundamental Research on Matter (FOM-TFF). It is also supported by the European Communities under the contract of Association between EURATOM and FOM and carried out within the framework of the European Fusion Programme.

Appendix A. Ion composition of an Ar/H 2 plasma

Analysis of an IV-characteristic measured by a double Langmuir probe by means of the Peterson and Albot theory requires the ion mass as input parameter [START_REF] Peterson | Collisionless electrostatic single-probe and double-probe measurements[END_REF]. The ion


John Wiley

Mónica Cozar

Roser Urreizti

Laura Vilarinho

Carola Grosso

Raquel Dodelson De Kremer

Carla G Asteggiano

Jaime Dalmau

Ana María García

María Antonia Vilaseca

Daniel Grinberg

Dr Susana Balcells
email: sbalcells@ub.edu

Identification And Functional Analyses of CBS Alleles In Spanish And Argentinian Homocystinuric Patients

Keywords: CBS mutations, homocystinuria, Southern blot, mini-gene, heterologous expression, enzyme activity CBS mutations, homocystinuria, Southern blot, mini-gene, heterologous expression, enzyme activity

Homocystinuria due to CBS deficiency (MIM# 236200) is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian.

Twenty-two different mutations were found, ten of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794bp deletion (c.532-37_736+438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in E. coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western-blotting. Mutations p.M173del, p.I278S, p.D281N and p.D321V showed null activity in all conditions tested, while mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions.

Introduction

Homocysteine (Hcy) is a sulfur-containing amino acid that occupies a major regulatory branch point in methionine metabolism. Homocysteine may be either remethylated to methionine or catabolized to form cysteine through the transsulfuration pathway. The first step of transsulfuration involves the condensation of homocysteine with serine to form cystathionine, a reaction catalyzed by the enzyme cystathionine β-synthase (CBS; EC 4.2.1.22).

Mutations in the CBS gene (MIM# 613381) cause classical homocystinuria (Hcu, MIM# 236200), an autosomal recessive disease characterized by severe hyperhomocysteinemia and homocystinuria, decreased plasma levels of cysteine and, often, hypermethioninemia. At the clinical level, classical homocystinuria mainly affects the eye, the skeleton, the vascular system and the central nervous system (CNS). Symptoms usually include ectopia lentis, osteoporosis, scoliosis, Marfanoid features, premature arteriosclerosis, thromboembolism and mental retardation [START_REF] Mudd | The natural history of homocystinuria due to cystathionine beta-synthase deficiency[END_REF]. Age of onset and disease severity are highly variable, ranging from dramatically affected children to asymptomatic adults [START_REF] Magner | Vascular presentation of cystathionine beta-synthase deficiency in adulthood[END_REF][START_REF] Skovby | A revisit to the natural history of homocystinuria due to cystathionine beta-synthase deficiency[END_REF][START_REF] Walter | Strategies for the treatment of cystathionine beta-synthase deficiency: the experience of the Willink Biochemical Genetics Unit over the past 30 years[END_REF][START_REF] Yap | Homocystinuria due to cystathionine beta-synthase deficiency in Ireland: 25 years' experience of a newborn screened and treated population with reference to clinical outcome and biochemical control[END_REF]. Treatments that lower tHcy, such as B-vitamins, dietary methionine restriction, and betaine supplementation, can significantly reduce the incidence of vascular events (the main cause of death in these patients) and improve the neurological problems [START_REF] Wilcken | The natural history of vascular disease in homocystinuria and the effects of treatment[END_REF][START_REF] Yap | Vascular outcome in patients with homocystinuria due to cystathionine beta-synthase deficiency treated chronically -A multicenter observational study[END_REF][START_REF] Yap | Homocystinuria due to cystathionine beta-synthase deficiency in Ireland: 25 years' experience of a newborn screened and treated population with reference to clinical outcome and biochemical control[END_REF].

Homocystinuria is a rare disease with variable incidence. While the worldwide incidence is estimated as 1/344000 born alive [START_REF] Mudd | Isolated persistent hypermethioninemia[END_REF], in Qatar it is 1/3124 [START_REF] El-Said | A common mutation in the CBS gene explains a high incidence of homocystinuria in the Qatari population[END_REF] and in Japan 1/800 000 [START_REF] Mudd | Isolated persistent hypermethioninemia[END_REF]. In Northern Europe the incidence may be of 1/20500 to 1/6400, as estimated from the high F o r P e e r R e v i e w 4 number of p.I278T mutation carriers found in some populations [START_REF] Gaustadnes | Prevalence of congenital homocystinuria in Denmark[END_REF][START_REF] Refsum | Birth prevalence of homocystinuria[END_REF].

To date, more than 150 different CBS mutations have been found worldwide, some of them common. Mutation p.I278T is considered panethnic and is particularly frequent in Northern Europe (Janosik, et al., 2001b;Kraus, et al., 1999;[START_REF] Moat | The molecular basis of cystathionine beta-synthase (CBS) deficiency in UK and US patients with homocystinuria[END_REF]. In Ireland, the p.G307S mutation accounts for 71% of the mutant alleles [START_REF] Gallagher | High frequency (71%) of Cystathionine B-Synthase Mutation G307S in Irish Homocystinuria Patients[END_REF], and in the Iberian Peninsula and Colombia, mutation p.T191M represents between 40% and 75% (Urreizti, et al., 2006a) of the mutant alleles.

A truncated form of the human CBS enzyme lacking the C-terminal regulatory domain has been crystallized (Janosik, et al., 2001a;Meier, et al., 2001) and structurally characterized [START_REF] Banerjee | Redox regulation and reaction mechanism of human cystathionineβ-synthase: a PLP-dependent hemesensor protein[END_REF][START_REF] Kabil | Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from 31P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme[END_REF][START_REF] Kery | Binding of pyridoxal 5phosphate to the heme protein human cystathionine beta-synthase[END_REF]Meier, et al., 2001;[START_REF] Taoka | Human cystathionine beta-synthase is a heme sensor protein. evidence that the redox sensor is heme and not the vicinal cysteines in the CXXC motif seen in the crystal structure of the truncated enzyme[END_REF]. The active form of this cytoplasmic enzyme is a homotetramer of four 63-KDa subunits. Each subunit combines one heme group and one pyridoxal phosphate (PLP), the latter acting as a cofactor in the reaction. In addition, each subunit binds the allosteric activator S-Adenosil-Methionine (S-AdoMet or SAM), an intermediate in the methionine cycle.

Heterologous expression in E. coli has been widely used to test the functionality of the CBS mutant alleles independently from the patient's genetic background [START_REF] De Franchis | Identical genotipes in siblings with differnt homocystinuric phenotypes: identification of three mutations in cystationine B-synthase usin en improved bacterial expression system[END_REF][START_REF] Katsushima | Expression study of mutant cystathionine beta-synthase found in Japanese patients with homocystinuria[END_REF][START_REF] Maclean | High homocysteine and thrombosis without connective tissue disorders are associated with a novel class of cystathionine beta-synthase (CBS) mutations[END_REF]. It has been proven to be a useful tool since the E. coli-expressed human CBS is indistinguishable from that obtained from cultured fibroblasts [START_REF] Bukovska | Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization[END_REF][START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: Homocystinuria due to Cystathionine β -Synthase Deficiency[END_REF]. In addition, in vitro, its relative activity in response to PLP and SAM is comparable to that of the enzyme obtained from human tissues [START_REF] Bukovska | Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization[END_REF][START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: Homocystinuria due to Cystathionine β -Synthase Deficiency[END_REF]. This study updates the CBS mutation spectrum of the homocystinuric patients from the Iberian Peninsula by presenting the analysis of 16 new cases. It also includes one Norwegian, one Indian and four Argentinian patients. In this cohort of 22 cases, 22 mutations were found, ten of which were novel. The new mutations include a deletion of 794 bp (c.532-37_736+438del794) detected by Southern blot analysis and an intronic deletion that leads to the skipping of exon 6, which was characterized using a minigene strategy. The pathogenic role of seven of the changes was assessed by heterologous expression of these mutant proteins, and their stability and activity were analyzed.

Materials And Methods

Patients

Twenty-six patients with classical homocystinuria from 22 unrelated pedigrees were involved in this study. Patients were initially diagnosed by their physicians on the basis of clinical manifestations suggestive of homozygous CBS deficiency.

Biochemically, these patients presented with a combination of severe hyperhomocysteinemia (typically above 150 µmol/l), and hypermethioninemia (typically above 40 µmol/l). Thirteen Spanish, three Portuguese, one Norwegian, one Indian and four Argentinian patients were included in the study. Our research was conducted in accordance with the tenets of the Declaration of Helsinki. The nature and possible consequences of the study were first explained to all patients and/or their parents, before their informed consent for inclusion in the research project was obtained. All 16 CBS coding exons (including exon 15) and their intronic flanking regions, were amplified by PCR and sequenced as described previously (Urreizti, et al., 2006a) with some modifications. Briefly, PCR reactions were performed on a final volume of 50 µl with 50 ng gDNA, 0.2 mM of each dNTP, 0.4 mM of each primer, 1.5-2.5 mM MgCl2 and 1.25 U of GoTaq ® Flexi DNA Polymerase (Promega, Madison, WI, USA). All mutations detected were confirmed by restriction analysis of the PCR products with the appropriate restriction enzyme, and the presence of all new mutations was assessed in 100 control chromosomes from Spanish anonymous donors. Primer sequences and PCR conditions have been described previously (Urreizti, et al., 2006a). MTHFR c.677C>T (rs1801133) was analyzed in all patients as described in Frosst et al. [START_REF] Frosst | A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase [letter][END_REF].

To characterize the deletion found in patient 87, genomic DNAs of the patient and her parents were PCR-amplified using primers 4F and 7R as described above with the addition of a final concentration of 5% DMSO. The PCR fragments were purified and sequenced.

For naming the mutations the following reference sequences were used: Genomic, GenBank NG_008938.1; cDNA, ENST00000352178. 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). The initiation codon is codon 1.

Southern blot

A Southern blot to analyze a 36-kb fragment of the CBS genomic region (Fig. 1), between two DrdI sites, encompassing all 23 exons, plus 6 kb of the 5' flanking region and 11.5 kb of the 3' region, was performed as follows: 10 µg of patient and control gDNA were double digested by DrdI and AflII (NEBiolabs, Ipswich, MA, USA), electrophoresed on a 0.9% agarose gel, blotted onto Amersham Hybond TM -N+ (GE Healthcare, Waukesha, WI, USA) membrane using standard protocols, and fixed by UV crosslinking. The DrdI/AflII double digestion of the 36 kb yields, in a wild-type setting, four fragments of around 15 kb, 10 kb, 8 kb and 3 kb, respectively (Fig. 1A).

Probe design, preparation and hybridization

Four probes were designed by selecting one unique sequence of approximately 600 bp within each restriction fragment. The sequences were aligned to the whole genome by BLAST, to confirm their specificity. These four sequences were PCRamplified from total genomic DNA (as described above), cloned into pUC19 vector (Fermentas, Burlington, ON, Canada) and sequenced. Probes were obtained from the clones by using digoxigenin-dUTP and the "PCR DIG Probe Synthesis Kit" (Roche, Mannheim, Germany), according to the manufacturer's instructions. Primer sequences for the probes are available on request. Labelled probes were subsequently purified using Illustra TM GFX TM PCR DNA and a Gel Band Purification Kit (GE Healthcare, Waukesha, WI, USA).

All subsequent steps of the Southern protocol (prehybridization, hybridization and developing of the filters) were performed using reagents (Dig Easy Hyb) and protocols (Dig Application Manual) from Roche (Mannheim, Germany).

Site-directed mutagenesis, heterologous expression and in vitro enzyme activity assays of the CBS mutations

All CBS mutant constructs were derived from the wild-type CBS expression plasmid pHCS3 [START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: Homocystinuria due to Cystathionine β -Synthase Deficiency[END_REF], a gift from the authors of that study. Each mutation was introduced into the wild-type expression plasmid using a Quik Change II XL TM Site-Directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, CA, USA) and expressed in XL-Gold E. coli cells as described in Urreizti et al. (Urreizti, et al., 2006b). Expression of pHCS3 was used as a positive control, and that of the empty vector pKK388.1 was included as a negative control. Protein extracts were obtained and CBS activity was measured as described in Kraus [START_REF] Kraus | Cystathionine b-synthase[END_REF] with some modifications (Urreizti, et al., 2006b).

CBS protein analysis

To assess the presence and relative amount of wild-type and mutant CBS proteins, Western blot analysis of the soluble fraction of the crude cell lysates was performed under denaturing conditions as described in Janosik et al. (Janosik, et al., 2001b), with some modifications (Urreizti, et al., 2006b).

Minigene construction and splicing assay

Genomic DNA from patient 68a (heterozygote for mutation c.667-14_-7del8) was amplified using CBS primers 4F and 6R to obtain a fragment of 626 or 618 bp (from the last 29 bp of intron 3 to the first 103 bp of intron 6). This PCR product, containing both wild-type and mutant alleles, was purified using Illustra TM GFX TM PCR amplification of each cDNA product was performed using primers T7 and SP6 as previously described [START_REF] Diaz-Font | Clinical and mutational characterization of three patients with multiple sulfatase deficiency: report of a new splicing mutation[END_REF][START_REF] Santamaria | SR proteins and the nonsense-mediated decay mechanism are involved in human GLB1 gene alternative splicing[END_REF]. The PCR products were electrophoresed and each band of interest was purified and analyzed by sequencing. 1), ten of them novel, were found in 22 unrelated patients of different geographical origin (Table 2). Forty-two mutant alleles were identified through sequencing of coding exons and flanking intronic regions, while an additional mutant allele was discovered by Southern blot analysis (see below). Only one allele remained unidentified. Five of the new mutations were missense while five were deletions.

Results

Screening for CBS mutations

After sequencing the complete CBS coding sequence, only mutation c.1566delG was found in heterozygosis in patient 87, which she inherited from her father. A Southern blot analysis of this patient and her mother, hybridized with a set of four probes covering the entire CBS genomic region (Fig. 1A), revealed the same pattern of bands observed in control samples but the relative intensities of fragments 2, 3 and 4 were altered (Fig. 1B). Hybridization of the same membrane separately with each individual probe revealed an additional band of nearly 10 kb detected by probes B and C (Fig. 1C). This result was consistent with a deletion removing the DrdI site present in intron 5. To test this hypothesis, a PCR amplification from intron 3 to intron 7 was performed. As shown in Fig. 1D, a new band of 1176 bp was observed in the samples of the patient and her mother. Sequencing revealed a 796-bp deletion (c.532-37_736+438del794), which spans from the last 37 bp of intron 4 to the first 438 bp of intron 6 (Fig. 1E). This deletion causes a frameshift starting from a valine to glycine substitution at position 178 and leading to a stop codon at residue 201 (p.V178GfsX23).

Analyses of pathogenicity of mutant CBS enzymes

None of the new mutations found was present in 100 control chromosomes, ruling out the possibility of common polymorphisms. We assessed the pathogenicity of all five new missense mutations and of the novel in-frame deletion p.M173del using an E. coli heterologous expression system followed by Western blot and in vitro enzyme activity analysis (Fig. 2). Mutation p.P49L, previously described by de Franchis et al. [START_REF] De Franchis | Clinical aspects of cystathionine beta-synthase deficiency: how wide is the spectrum? The Italian Collaborative Study Group on Homocystinuria[END_REF] was also analyzed.

All the proteins from the mutant alleles were found in amounts similar to those of the wild type (Fig. 2, upper panel). The activity of these mutant enzymes was assayed in vitro, either in the presence or absence of the cofactor PLP, or in the joint presence of PLP and SAM (Fig. 2, lower panel). As previously described [START_REF] Kluijtmans | Defective Cystathionine B-synthase regulation by S-Adenosylmethionine in a partially pyridoxine responsive homocystinuria patient[END_REF][START_REF] Maclean | High homocysteine and thrombosis without connective tissue disorders are associated with a novel class of cystathionine beta-synthase (CBS) mutations[END_REF], we found that the wild-type CBS activity was strongly stimulated by both PLP and the combination of PLP and SAM. Mutations p.M173del, p.I278S, p.D281N and p.D321V showed null activity, either in the absence or presence of the activators. In contrast, mutations p.49L, p.P200L and p.A446S retained different amounts of activity and also the ability to be stimulated.

In particular, mutation p.P49L displayed null activity in the absence of any of the activators, retained 71% of the wild-type activity in the presence of PLP and showed reduced activation by SAM. In contrast, in the absence of any of the activators, mutation p.P200L displayed around a third of the activity of the wild type (15% versus 43%) and retained the ability to be induced by PLP and SAM. SAM stimulation was 2-fold, comparable to that of the wild type. Finally, p.A446S displayed highly variable levels of activity in different tests, in the absence or presence of activators, but in general the values where in the range of the wild type or higher. The small deletion c.667-14_-7del8 involved intron 5 sequences within the acceptor site, without affecting the conserved AG dinucleotide. Since no RNA was available from patients with this mutation (68a and 68b), a minigene assay was performed to test the effect of this mutation on RNA splicing (Fig. 3A). The genomic region spanning from the 3' part of intron 3 to the 5' part of intron 6 of the CBS gene of the mutant and wild-type alleles was PCR-amplified and cloned within intron 7 of a construct containing several GLB1 exons (see Material and Methods for details).

Effect of a small intronic deletion on RNA splicing

Wild-type and mutant constructs were transfected into HeLa cells and splicing was assayed by RT-PCR using the T7 and SP6 primers (Fig. 3B). The major band observed in the wild-type sample corresponds to the inclusion of all three CBS exons, whereas in the mutant, exon 6 was skipped, as assessed by sequencing of all the bands.

Discussion

Mutation p.T191M was identified in 21.8% of the alleles of the Spanish and Portuguese patients included in this study (Table 2). Taking into consideration all 50 homocystinuric patients from the Iberian Peninsula included in this and in our previous studies [START_REF] Urreizti | Spectrum of CBS mutations in 16 homocystinuric patients from the Iberian Peninsula: high prevalence of T191M and absence of I278T or G307S[END_REF]2006a), mutation p.T191M accounts for 44% of the mutant chromosomes. Mutation c.1566delG is the second most prevalent change (7% overall), and is particularly common in Portugal (21%). In the present study, we found the panethnic mutation p.I278T for the first time in two unrelated Spanish patients, both in the heterozygote state, and in a Norwegian patient, in homozygosis (Table 2). Missense mutations are the most common alterations in the CBS gene in the patients of the present study (73%). We also found six deletions of different sizes (1 bp to 794 bp), five of them novel. An intronic 8-bp deletion (c.667-14_-7del8) was found in two siblings (# 68a-# 68b, Table 2). Prediction algorithms indicated a subtle reduction in the score of the splicing acceptor site of intron 5. However, a mini-gene analysis clearly showed that it led to the skipping of exon 6, and produced a frameshift. This is consistent with the severity of the patients' phenotypes. We also found a 5 bp deletion in exon 8 in patient # 109, and a 1 bp deletion in exon 6 in patient # 89, both in homozygosis (Table 2). The severity of these genotypes is in accordance with the severe clinical presentation of both patients. In general, frameshift-generating deletions correlate with a more consistently severe phenotype, as expected.

Seven mutations (six missense and a 3-bp deletion) were expressed in E. coli and analyzed by Western blot under denaturing conditions (Fig. 2, upper panel).

Their presence in amounts similar to those of the wild-type CBS suggests that the mutations do not affect protein integrity. Alternatively, they may affect the catalytic core, proper protein folding or the ability to form tetramers. In this regard, several authors have studied the involvement of certain chaperones in the rescue of missense CBS mutants, and demonstrated that the regulation of these chaperones could lead to almost complete recovery of the mutant protein activity (Kopecka, et al., 2010;[START_REF] Majtan | Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes[END_REF][START_REF] Singh | Chemical chaperone rescue of mutant human cystathionine beta-synthase[END_REF][START_REF] Singh | Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70[END_REF].

Four mutations showed null activity, which supports their pathogenic character. These mutations, p.D281N, p.I278S, p.D321V and p.M173del, were found in four unrelated patients, # 82a (and his siblings), # 94, # 90, and # 55, respectively. No correlation between their null activity and the phenotypes of the patients could be established since all but patient # 94 presented with mild disease.

The other three mutations, displaying different degrees of enzyme activity, were p.P49L, p.P200L and p.A446S, present in patients # 80, # 79 and # 90, respectively, all in the heterozygous state. In these cases, a correlation between their residual activity and the mild phenotype of the patients was observed.

We found the activity of p.P49L to be 75% of that in the wild type. While our work was ongoing, Kozich et al. [START_REF] Kozich | Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity[END_REF] published an analysis of mutation p.P49L expressed in E. coli. They found it to be an active enzyme, indistinguishable from the wild type. Mutation p.P49L was found in two siblings, one with adult onset and the other asymptomatic. Genetic analysis of the proband (# 80a in Table 2 and subject II.1 in Fig. 4), showed compound heterozygosity for p.P49L and p.R125Q (Table 2). Genotyping of his siblings revealed an asymptomatic sister also carrying both mutations (# 80b; II.3 in Fig. 4). The proband had suffered a myocardial infarction at age 53. Blood testing revealed moderate hyperhomocysteinemia and hypermethioninemia (58 and 655 µM, respectively) and he responded well to PLP (tHcy: 28 µM after treatment). On the other hand, his asymptomatic sister, aged 54, presented with severe hyperhomocysteinemia (543 µM) and hypermethioninemia (1723 µM) but had no clinical sign of classical homocystinuria and had given birth to three healthy offspring without complications. As the two CBS mutations were inherited separately by the offspring, we know that they were not in cis in the mother or in his affected brother. Mutation p.R125Q, the other mutation in family # 80, was originally described as a null activity mutation [START_REF] Sebastio | The molecular basis of homocystinuria due to Cystathionina B-Sinthasa deficiency in italian families, and report of four novel mutations[END_REF]. In their recent publications, Kozich et al. [START_REF] Kozich | Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity[END_REF] and Majtan et al. [START_REF] Majtan | Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes[END_REF] expressed it in E. coli. In both studies the reduced activity was improved under permissive conditions or in the presence of chaperones. Taken together, these new results on p.P49L and p.R125Q help explain the mild or null affectation of patients # 80a and # 80b. In addition, the MTHFR 677C>T polymorphism may act as a phenotype modifier in this family. However, the discrepancy between the blood markers and the phenotype of patient # 80b remains unexplained.

Mutation p.P200L was identified as the sole mutation in patient # 79. The patient's symptoms are limited to hypertension and severe hyperhomocysteinemia (183 µM), which does not respond to vitamin B6 and folate treatment. A second CBS mutation may be deep intronic, located in a regulatory region far from the gene, or involved in a complex rearrangement not detected by our screening methods. Mutation p.A446S was found in a mildly affected patient (# 90, Table 2), in compound heterozygosis with the null mutation p.D321V (Fig. 2). The activity of p.A446S was very similar to that of the wild type and this correlates with the mild phenotype of the patient, who only presented with lens dislocation at 45 years, despite a high tHcy level (105 µM, which was reduced to 40 µM after two months of treatment with folic acid and vitamins B6 and B12).

The poor genotype-phenotype correlation widely observed by us and others among the homocystinuric patients and the demonstration that some patients Novel missense mutations were checked in control chromosomes using the indicated enzyme. +: the mutation creates a new site; -: the mutation destroys a site. Reference Sequences: Genomic, GenBank NG_008938.1; cDNA, ENST00000352178. 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). The initiation codon is codon 1.

  prepared from peripheral blood leukocytes, using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA).

  a Gel Band Purification Kit (GE Healthcare, Waukesha, WI, USA) and cloned into the pGLB1 vector[START_REF] Diaz-Font | Clinical and mutational characterization of three patients with multiple sulfatase deficiency: report of a new splicing mutation[END_REF]. Plasmid pGLB1 is based on a pcDNA3.1 vector and contains exons 7, 8, and 9 and introns 7 and 8 of the GLB1 gene, where intron 7 contains a PmeI restriction site, which was used to clone the CBS fragment. The resulting plasmids, named pGLB1-CBS_wt and pGLB1-CBS_mut, were confirmed by sequencing.The splicing assay was performed by transfecting 1 µg of each minigene plasmid with 5 µl of Lipofectamine 2000 Reagent (Life Technologies, Basel, Switzerland) into 90% confluent HeLa cells. Total RNAs were isolated 24 h after transfection using the QIAshredder and the RNeasy Mini Kit (QIAGEN, Hilden, Germany). RNA concentration was determined spectrophotometrically, integrity was verified by gel electrophoresis and RNA quality was assessed by OD 260/230 and 260/280 ratios.Two µg of each RNA was reverse-transcribed in a final volume of 20 µl using a High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

  different mutations (Table

  allowed us to identify a large deletion of nearly 800 bp in the heterozygous state p.V178GfsX23) in patient 87 and in her mother. Additionally, a qmfPCR assay was performed on the patient's sample yielding a consistent result (C. Ged, personal communication). This patient, diagnosed through neonatal screening, presented with dramatically increased plasma methionine (700 µM) and total homocysteine (145 µM). She has been on a normal diet and treated with pyridoxal, folate and betaine since her birth. Currently aged 4 years, her plasma homocysteine and methionine levels are 76 µM and 302 µM, respectively, and she is clinically asymptomatic, highlighting the importance of an early diagnosis.

Figure 1 .

 1 Figure Legends:

Figure 2 .

 2 Figure 2. Heterologous expression and activity of seven CBS mutations. A: SDS-PAGE followed by Western blot immunodetection of wild-type and mutant CBS

Figure 3 .

 3 Figure 3. Splicing analysis of mutation c.667-14_-7del8 included in a minigene construction. A: scheme of the minigene: grey boxes represent GLB1 exons and white boxes are CBS exons. The thick horizontal line represents the CBS introns and the thin line the GLB1 introns. PCMV: CMV promoter; BGHpA: BGH polyadenylation site. Wild-type and mutant splicing patterns are indicated above and below the gene respectively. The dot indicates the position of the mutation. The wild-type and mutant constructs were transiently transfected into HeLa cells. Total RNA was extracted 24 h after transfection, reverse-transcribed and PCR-amplified with primers T7 and SP6 (indicated by arrows). B: PCR amplification of the wild-type (1) mutant (2) and the minigene construct without the CBS exons (3). The asterisk points to a minor band lacking exon 4 observed in the wild-type construct. C: Diagrams showing the three main PCR products.

Figure 4 .

 4 Figure 4. Pedigree of family 80. Patient 80a (II.1), the proband, is marked with an

Table 1 . List Of The 22 Different Mutations Found In This Cohort.

 1 Castro et al., 1999 Mutations not previously described are indicated in bold 1

	DNA	Deduced Protein Change	Exon-Intron	Alleles	Restr. Enz. 1	References
	c.146C>T	p.P49L	Ex 1	1		de Franchis et al., 1998
	c.253G>A	p.G85R	Ex 2	1		Maclean et al., 2002
	c.361C>T	p.R121C	Ex 3	1		Katsushima et al., 2006
	c.374G>A	p.R125Q	Ex 3	1		Marble et al., 1994
	c.518delTGA	p.M173del	Ex 4	2		This study
	c.532-37_736+438del794 p.V178GfsX23 In 4-In 6	1		This study
	c.572C>T	p.T191M	Ex 5	7		Urreizti et al., 2003
	c.599C>T	p.P200L	Ex 5	1	MspI-	This study
	c.667-14_667-7del8	p.Y223GfsX23	In 5	1		This study
	c.676G>A	p.A226T	Ex 6	2		Shan et al., 1998
	c.689delT	p.L230RfsX39	Ex 6	1	MspI+	This study
	c.824G>A	p.C275Y	Ex 7	1		Urreizti et al., 2003
	c.833T>G	p.I278S	Ex 8	1	TspRi+	This study
	c.833T>C	p.I278T	Ex 8	4		Kozich et al., 1992
	c.841C>T	p.D281N	Ex 8	1	BamHI-	This study
	c.862_866del5	p.E289GfsX39	Ex 8	2		This study
	c.962A>T	p.D321V	Ex 9	1	Hpy8I-	This study
	c.1085C>T	p.T353M	Ex 10	2		Dawson et al., 1997
	c.1136G>A	p.R379Q	Ex 10	1		Urreizti et al., 2003
	c.1330G>A	p.D444N	Ex 12	4		Kluijtmans et al., 1996
	c.1336G>T	p.A446S	Ex 12	1	BstNI+	This study
	c.1566delG	p.K523SfsX18	Ex 16	5

Table 2 . Patients, CBS And MTHFR C.667C>T Genotypes, Disease Severity And Geographical Origin

 2

			MTHF
			R
			(c.677
	Patient	Genotype	C>T)	Severity	Country
	74	p.[T353M]+[D444N]	TT	Severe	Spain
	79	p.[P200L]+[?]	CT	Asymptomatic	Spain
	80a	p.[R125Q]+[P49L]	TT	Mild	Spain
	80b	p.[R125Q]+[P49L]	CT	Asymptomatic
	82a	p.[T191M]+[D281N]	CC	Mild	Spain
	82b	p.[T191M]+[D281N]	CC	Mild
	82c	p.[T191M]+[D281N]	CC	Mild
	83	p.[T353M]+[D444N]	TT	Mild	Spain
	84	p.[I278T]+[R121C]	CT	Mild	Spain
	88	p.[D444N]+[D444N]	CT	Mild	Spain
	92	p.[T191M]+[T191M]	CT	Mild	Spain
	93	p.[T191M]+[T191M]	CT	Severe	Spain
	94	p.[T191M]+[I278S]	CT	Severe	Spain
	100	p.[T191M]+[R379Q]	CT	Mild	Spain
	107	c.[1566delG]+[1566delG]	CT	NA	Spain
	108	p.[I278T]+[C275Y]	TT	Mild	Spain
	81	c.[1566delG]+[1566delG]	TT	Severe	Portugal
	87	c.[1566delG]+[532-37_736+438del794]	CC	neonatal screening	Portugal
				(asymptomatic)
	109	p.[E289GfsX39]+[E289GfsX39]	CT	Severe	Portugal
	68a	p.[G85R]+c.[667-14_667-7del8]	CT	Severe	Argentina
	68b	p.[G85R]+c.[667-14_667-7del8]	CT	Severe
	89	c.[689delT]+[689delT]	CT	Severe	Argentina
	90	p.[D321V]+[A446S]	CT	Mild	Argentina
	91	p.[A226T]+[A226T]	CT	Severe	Argentina
	105	p.[I278T]+[I278T]	ND	NA	Norway
	55	p.[M173del]+[M173del]	ND	Mild	Indian
	ND: Not Determined.
	NA: Not Available.

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

The authors are grateful to the patients and their families. We also thank the following colleagues for providing samples and clinical data: A.Baldellou and M.T.Calvo (Zaragoza), M.T.García-Silva and M.Pardo (Madrid), M.Urtasun (San Sebastian), O.Alonso (Huelva), A.Reparaz and C.Torreira (Vigo), I.C.Verma (New Delhi, India). Robin Rycroft revised the English. The CIBER de Enfermedades Raras (CIBERER) is an initiative of the ISCIII. This study was supported by grants from Ministerio de Ciencia e Innovación (Grants SAF2007-64654; SAF2010-15707; PIB2010AR-00473), and the Catalan Department of Universities, Research and the Information Society (2005SGR 00848, 2009 SGR 971).