Primary Teachers' Particle Ideas and Explanations of Physical Phenomena: The Effect of an In-Service Training Course

Keywords: teacher knowledge, science education, in-service, primary school, misconception user): Particle ideas, Physical phenomena Primary teachers, Particle ideas, Physical phenomena, In-service training

This paper presents a study concerning Greek primary school teachers' (n=162) ideas about the particulate nature of matter and their explanations of physical phenomena. The study took place during an in-service training course where the effectiveness of a specially designed intervention was tested. A key feature was an approach based on the concept of a substance and its states rather than 'solids, liquids and gases'. Pre-intervention, the teachers held misconceptions similar to those of pupils. Also, there seemed to be some relationship between the teachers' particle model ideas and their explanations of phenomena. Postintervention, the teachers' descriptions and explanations were found to be significantly improved, with almost zero correlation between pre and post intervention scores.

Implications for science education are discussed.

Introduction

The late 1980s saw a shift from a largely process approach to a greater emphasis on subject knowledge for primary school science in the USA, UK, Australia and New Zealand [START_REF] Osborne | Primary science: past and future directions[END_REF]. At that time, the Greek primary science curriculum was reconstructed to also feature content more prominently (Greek Ministry of National Education and Religion Affair, 1985). These curricular changes placed demands on in-service primary teachers for which few were well qualified, particularly in the physical sciences. Indeed, matter (Author#3, 1996) and hence there must be three types of particle which carry the respective macroscopic properties, a common misconception (e.g. [START_REF] Andersson | Pupils' conceptions of matter and its transformations (age 12-16)[END_REF]Ben-Zvi, Eylon, & Silberstein,1986;[START_REF] Gabel | Understanding the particulate nature of matter[END_REF][START_REF] Griffiths | Grade-12 students' misconceptions relating to fundamental characteristics of atoms and molecules[END_REF][START_REF] Kokkotas | Teaching the topic of the particulate nature of matter in prospective teachers' training courses[END_REF][START_REF] Pereira | Pupils' representations of models of water[END_REF]. Changes of state in some instances are then seen as perplexing anomalies. We advocate an approach which focuses on the concept of a substance and its states. Furthermore, we would argue that particulate ideas are necessary to sharing the scientific view of events on a macroscopic scale. For example, without identifying a substance with a kind of particle how can one accept that the substance water can be part of the clear air -be there to separate out on cooling the mixture? (Pupils seem to find the appearance of condensation on a cold object one of the most difficult events to understand.).

If particle theory needs to be an integral part of teaching about physical changes one must ask whether this package is appropriate for the primary curriculum. However, if the standard approach to introducing the particulate view of matter is flawed we cannot say yet what pupils could achieve otherwise at various ages. Using our new conceptual approach, short intervention studies with primary aged pupils have yielded encouraging results (Authors #1 & #3, 2005;Authors #1, #3 & other, 2008;Author#3 & #1, 2008). If primary pupils can engage with particle ideas, their teachers ought to be capable. However whether this can be realised in practice remains an empirical question, too. At older ages, teachers who have held misconceptions throughout their school years and teaching careers may be resistant to change -a widely noted characteristic of 'misconceptions'.

In this paper we report the outcome of an in-service course, which adopted a substance approach to the particle model and physical changes, for Greek primary teachers. In this context the following two research questions were addressed:

• What was the affect of the course on teachers' understanding of the particle model and their explanations of physical phenomena during this course? • What is the relationship between the degree of teachers' understanding of the particle model and their explanations of physical phenomena?

In both questions, commonalities with pupils' thinking were also examined.

The content and structure of the training course

The study took place during an in-service teachers' professional development Table 1 outlines the content covered by the course with respect to physical changes (further work on chemical change is not reported here). Similar content has been used with Greek primary pupils (Authors #1 & #3, 2005;Authors #1, #3 & other, 2008). The rational behind the scheme is discussed in full elsewhere (Author#3 & #1, 2008) and we give just a brief overview here.

[Insert Table 1

about here.]

Given the expected weaknesses in the teachers' background knowledge, the scheme starts by identifying properties which depend on the material only. Here material means any recognizable kind of 'stuff' be it a substance (element or compound) or a mixture of substances. Next, melting behaviour is used to give an operational definition of a substance: a sample of stuff which has a precise melting point (there is a sharp change between solid and liquid) as opposed to a mixture which melts over a temperature range. The particulate view of matter is then introduced to explain why a substance can change between the solid and liquid states. At the outset, the intention is to counter any suggestions that 'solids' and 'liquids' are two types of matter. A simple model is presented which talks about the 'particles of a substance', where the identity of the substance rests with the particles: different substances are different particles (there is no distinction between molecules, atoms or ions). These particles have an 'ability to hold' on to each other (which is different for different substances) and an energy of movement (which depends on the temperature). The 'ability to hold' is a characteristic of a substance and does not change on change of state. Melting is explained by increased energy of movement which partially overcomes the hold so the particles can move around from place to place, but are still kept close together. The particles themselves, whatever their physical nature, do not change. Following on, the change to the gas state (boiling) is explained by the particles having enough energy to completely overcome the 'hold'. Crucially, the coexistence of different substances in different states at the same temperature is explained by differences in the 'ability to hold' (which give different melting and boiling points). Substances in the gas state at room temperature have a very weak hold between their particles -they are not a different kind of matter. (NB: We prefer to use 'hold' rather than attraction since 'hold' anticipates later refinement into the idea of a bond as a balance between attraction and repulsion.) Finally, the idea of a distribution of energy amongst particles at a particular temperature is introduced to explain evaporation of water into the air at room temperature, and the separation of water from the air on cooling.

Methodology

Sample

One hundred and sixty two primary school teachers (68 male and 94 female) participated in the present study. All were working in primary schools in the area of East Macedonia and Thrace, Greece, and their teaching experience ranged from 2 to 21 years (mean 12.7 and SD=4.5). The teachers had not participated in any similar in-service training programs previously. Their undergraduate studies on general science had been the only formal education on this matter. During their in-service teaching they would have acquired similar experiences, since all would have been using the same mandated textbook and guidance (Greek Pedagogical Institute, 2003). It should be stressed here that teaching experience does not necessarily enhance conceptual learning or bring expertise in science teaching [START_REF] Morrison | Science teachers' diagnosis and understanding of students' preconceptions[END_REF].

Teaching

The teaching took place in nine independent locations, each with a class of 18 participants. The whole course consisted of 30 one-hour lessons; six covered sections 1 to 3 of Table 1 (the particulate nature of substances) and nine covered sections 4 and 5 (explanations of corresponding physical phenomena). The remaining lessons covered chemical change (not reported here). All of the teaching was carried out by the first author.

Simple demonstration experiments (e.g. melting of wax, melting of chocolate, boiling of water) were used in the lessons. For each of these, 2-3 teachers were invited to participate (handling materials, heating etc.), while the rest observed. Overall, most of the teachers in each class did not volunteer, saying that they didn't feel comfortable with such experiments and preferred to watch. Only around 4-8 teachers in each class had direct involvement in one or more these demonstrations. For more difficult or time-consuming experiments, appropriate videos were used (e.g. evaporation of alcohol or water, boiling of wax). All teachers participated in discussions on the observations and explanations. Static representations concerning the particulate nature of substances were used as aids. Part I contained two groups of tasks -five tasks in total. The groups correspond to two dimensions in which the degree of the development of particle ideas can be measured, namely, the Particulate/ Continuous dimension and the Macroscopic/ Collective dimension.

These dimensions had emerged from a longitudinal study on pupils' understanding of the particulate view of matter (Author #3, 1998a). The former concerns the development of the notion of particles in terms of the relationship between the particles and the substance. At one end a completely continuous view of matter is held, at the other the particles are the substance. Between these two ends, the particles are embedded in the substance with varying degrees of association between the particles and the substance. The macroscopic/collective dimension relates to the association between the particles and the macroscopic properties of a state. At one end all of the macroscopic properties are carried by each individual particle. At the other end, the macroscopic properties are explained by the collective behaviour of the particles (and the physical nature of individual particles is irrelevant).

Part II consisted of four tasks, which concern measurements on the description and explanation of physical phenomena: melting, boiling, evaporation and condensation.

Descriptions of all the instrument tasks for Parts I and II are given in Table 2. Figure 1 gives some of the representations used. A month after the training course, the same instrument was used as a post-intervention test. The total time period for the completion of the tests was one hour (60 min) each.

[Insert Table 2 about here] and [Insert Figure 1

Data analysis

As the instrument was a written test applied to a large sample the data analysis is mainly quantitative. Qualitative characteristics of answers were used for categorization, but are not reported and discussed in detail. In each one of the tasks, teachers' answers were categorized according to their correctness by two of the authors (the 1 st and the 2 nd ). The agreement percentage after discussion and negotiation became 100%. Depending on the task, a teacher's answer could be characterized as: correct (C), partially correct (PC), not entirely incorrect (NEI) and incorrect (I). The corresponding scores awarded for these categories were 3 to 0, respectively. In some tasks the categorization resulted in three levels: correct, partially correct and incorrect and the scores awarded were 2 to 0, respectively. Total scores were calculated as sums and were used as percent achievement for further statistical analysis.

The validity of the test concerns the evaluation of achievement in a specific domain and thus it refers to content validity (Mertens, 2005, p 354). Thus, the establishment of this type of validity was based on elaborated judgement and expertise. The Cronbach's alpha was 0.69 and 0.59 for pre and post intervention questionnaires, respectively. Although not very high, they are satisfactory as indicators for internal consistency. Values down to 0.55 are acceptable for statistical consideration in social science studies [START_REF] Hatcher | A step-by-step approach to using the SAS system for univariate and multivariate statistics[END_REF]. However, the low Cronbach's α values in this case are not definitely related to lack of internal consistency. [START_REF] Kline | The handbook of psychological testing (2nd edition)[END_REF] notes that values below 0.7 can be expected in cognitive tests due to the diversity of constructs being measured.

Results and Discussion

In this section the results of the pre-and post-intervention tests are presented, discussed and compared with previous research findings. Parts I and II are considered separately and then in relation to each other through statistical analysis. 2, tasks 1-3) are presented in Table 3.

[Insert Table 3 about here] Pre-intervention, although the majority of the teachers had some ideas about particles (PC and NEI), few had a complete particulate picture of substances for the solid, liquid and gas states (C). The critical factor here was the idea of empty space (vacuum) between the particles. Teachers in the PC category did not provide any clarification on this. For NEI, responses indicated continuous matter around the particles. A quarter of the teachers expressed no ideas about particles. Post intervention the distribution changes and the numbers of teachers who gave correct answers increase significantly for all physical states. The numbers of incorrect views have almost disappeared.

Given the differences in age and cognitive maturity a comparison between success rates for teachers and pupils would not be reasonable. However, compared to our pupil interventions there is an interesting difference in trends. In both pre and post tests, teachers' particle ideas varied from the solid to gas states in a different way to younger pupils. The teachers' resistance in accepting particle ideas is stronger for the solid state than the gas state; the difference is statistically significant [χ 2 = 226, p<0.000]. The opposite seems to hold true for pupils (Authors #1 & #3, 2005;Author #3, 1998a), who have more difficulty in accepting particle ideas for the gas state. The idea of 'nothing' between particles seems to be especially challenging for pupils. Macroscopically, pupils accept the solid and the liquid states, perhaps because they can see and feel them, but not the gas state, which is invisible. Thus, possibly it is easier for them to apply new ideas to something more familiar, like the solid state. With more macro-experiences the gas state may not be so mysterious for the teachers. Due to their education, teachers are likely to be more aware of the different properties of the three states, such as the compressibility of gas state. As a result they find it easier to conceive of a vacuum between the particles when the spaces are large. [START_REF] Gomez | Evaluation of the degree of Coherence found in students' conceptions concerning the particulate nature of matter[END_REF], report that the understanding of the high compressibility of gas state (compared to that of liquid state) increases with the conceptualization of the particulate nature of matter and is related to age (for ages 9 to 22).

For pupils aged 11 -14 years old, Author #3 (1998a) found that improvement in the Macroscopic/Collective dimension was more difficult than in the Continuous/Particulate dimension. Tasks 4 and 5 (Table 2) explored the macroscopic-collective dimension. First, teachers were asked to describe and compare particles of the same substance (water) in different states (task 4). For task 5 teachers were asked to go a little further and to do the same for different substances (sugar, water and oxygen) in different states (at room temperature).

Here, the 'size' and 'shape' of particles, in addition to 'distance between particles' and 'motion' of particles, should be under consideration.

Table 4 shows the categories and frequencies of teachers' responses to the two tasks.

Pre intervention, 40.7% were in category I for both 4a and 4b. These teachers clearly ascribed macroscopic characteristics to the particles; i.e. solid, liquid and gas particles for the three states, respectively. Post intervention, incorrect responses dropped to 7.4% of the teachers.

From 7.3% pre-intervention, the percentage of teachers answering correctly (for both 4a and 4b) rose to 53.7%.

For task 5, pre-intervention, 46.3% were in category I for both 5a and 5b. These teachers did not note differences in size and shape. Again, macroscopic properties were emphasised; i.e., solid particles for sugar, liquid particles for water and gas particles for oxygen. Post-intervention, a significant number, 19.8%, remained in category I for both 5a and 5b. At 6.2% pre and 11.7% post intervention, the number in the correct category (for both 5a and 5b) was much lower than for task 4. Comparing Tables 3 and4, overall, the trend is similar to those we have found with pupils (Author #3, 1998a;Author#1 & #3, 2005). The macroscopic-collective dimension appears to be more difficult than the continuous-particulate [Insert Table 4 about here] Part II: Describing and explaining phenomena Tables 5 and6 present categories and frequencies of the teachers' responses to the four phenomena addressed in Part II of the test. There are two components for each phenomenon: the description of the event and the explanation.

Melting

Initially, most of the teachers described the liquid as still being wax (category C). Small numbers were either not definite in their answer (e.g. 'it is a liquid'-category PC) or referred to different names (e.g. it is water -category I). Neither of these necessarily implies that melted wax is being considered as a different substance. The former is a general description and the latter could be using 'water' as a 'prototype' for the liquid state. Indeed, the concept of a substance itself is not always clear for teachers (Author #1 et al., 2000) and the non-conservation of substance identity seems to go against common sense (Author #3, 2000(Author #3, , 2002)). However, relatively few (28 -17.3 %) gave acceptable particle structures for the solid and liquid states in their explanations. Post-instruction, all but one clearly identified the liquid as wax and 146 (90.1%) gave a 'correct' particle explanation.

[Insert Table 5 about [START_REF] Paik | K-8 th grade Korean students' conceptions of 'changes of state' and conditions for changes of state[END_REF], most of the teachers gave 'alternative' ideas when asked about the content of the bubbles in boiling water. The most frequent response was 'air' (39%, category NEI). Some caution in interpretation is needed here since 'air' could be a general term for the gas state (Author#3 & Other, 1996). For Greek people this might have additional reason to hold true since the words 'air' and 'gas' are alike in Greek language: air is 'aeras' and gas is 'aerio'. For category I, 26% suggested either oxygen or hydrogen or both. Rather than some kind of separation, in some cases this could reflect a lack of distinction between the concepts of mixture (oxygen and hydrogen) and chemical compound (water, a compound of oxygen and hydrogen). Whatever some teachers might have meant, only 25% gave the 'simple' answer that the bubbles were water in the gas state (water vapour was accepted). Postintervention, the ideas concerning 'oxygen and /or hydrogen' were abandoned, but 15% persisted with 'air'. Most were in the correct category (68%).

To explore the changes in structure between the liquid and gas states, the teachers were asked to give particle pictures for three areas: inside boiling water (liquid state), inside the bubbles (gas state) and just above the upper surface (where water is still in the gas statenot where condensation would be seen). Pre-intervention, results show teachers having significant problems with the structure in each area. Misconceptions noted in Part 1 (Table 3) seemed to impact on their answers here (Table 5). Since most teachers assigned 'a gas' of some kind to the content of the bubbles (i.e., water vapor, air or some other gas -task 7a) it might be expected that the percentage giving a gas structure for inside a bubble would be high. However, only 50% of the teachers, pre-intervention, gave the correct structure for the bubble. Interestingly, 40% of these (20% of the total) did not give a correct gas state structure for the area just above the upper surface of the liquid. This indicates that these teachers had not understood a crucial part of the mechanism of the boiling: i.e., that the structure just above the upper surface is the same as the bubbles since this is where the content of the bubbles is released. Post-intervention the overall situation shows a statistically significant (p<0.000) improvement. In relation to the gas state, 91.2% of the teachers gave the correct structure for the bubbles and only 7.4% of these could not give the correct structure for just above the upper surface as well.

Evaporation

The difference between boiling and evaporation at room temperature can be described at the macroscopic level and the particulate level. As the particulate level is examined in tasks 7a,b and 8b, the categorization in 8a considered the macroscopic level. For a correct response to 8a, answers were expected to address two criteria, explicitly or implicitly: i) that boiling takes place at a specific temperature whereas evaporation takes place over a range; ii) that the change takes place within the body of the liquid for boiling, whereas for evaporation it takes place at the surface. Pre-intervention, 43.2% of the teachers used both criteria for the distinction between boiling and evaporation satisfactorily (category C) and 29% addressed one (category PC). Nearly all of the teachers in the latter category referred to the temperature.

Post-intervention, 91.4% fall in category C.

Pre-intervention, very few teachers could use particle ideas to explain evaporation at room temperature (task 8b). Only 6 cases (0.3%) approached scientific thought (category C) using elements and terms from kinetic theory. These answers referred to a distribution of energy and the escape (individually) of high energy particles to mix in with the air particles.

Those placed in category PC (0.4%) implied this process, but without reference to energy distribution and the mixing with air particles. The majority (81%) of teachers gave insufficient general descriptions (NEI). Many of these were tautologies (e.g. 'evaporation happens when a liquid evaporates', or 'evaporation happens when a liquid goes to the gas state'). Thus far, the teachers' formal science education (at least in Greece) seems to have [Insert Table 6 about here]

Condensation

In contrast to pupils (e.g. Authors #1 & #3, 2005;Author #3, 1998c;Bar & Travis, 1991;[START_REF] Hatzinikita | Pupils' ideas on conservation during changes in the state of water[END_REF][START_REF] Paik | K-8 th grade Korean students' conceptions of 'changes of state' and conditions for changes of state[END_REF], the majority of the teachers seemed to appreciate that droplets appearing on a cold surface are water. Preintervention, 68% gave the correct answer, rising to 91% post-intervention. This could also be related to their richer everyday experiences compared to those of pupils. However, explanation is more problematic. Both, before and after the course, the large majority were in the PC category, 80% and 77% respectively. Here, responses acknowledged the role of the decreased temperature but went no further. Only one teacher pre-intervention and 22 post are in category C. This required links between the temperature decrease, loss of particle energy and the grouping together of (water) particles to give droplets in the liquid state. In comparison to pupils (e.g. Authors #1 & #3, 2005; Author #3, 1998c; [START_REF] Paik | K-8 th grade Korean students' conceptions of 'changes of state' and conditions for changes of state[END_REF], notwithstanding the teachers' greater comfort with the gas state (Part I) and their appreciation that the condensation is water, they also seem to have difficulty in explaining the phenomenon. As with evaporation at room temperature, condensation of atmospheric water seems to need further attention.

Statistical Analysis

The effect of the development of particle ideas

The relationships between particle model scores in Part I and description/explanation of phenomena scores in Part II were tested by correlation analysis. Treating the 7 gives the calculated Spearman's rho correlation coefficients.

[Insert Table 7 about here] Pre-intervention for the continuous/particulate dimension, correlation coefficients range from 0.16 to 0.58 and are all statistically significant. For the macroscopic/ collective dimension the correlations are generally lower and are not significant for melting and condensation. The latter lower correlations are not unexpected. Teachers holding weak particle models, which do not regard the particles as being the substance, would not necessarily think there would be any differences between individual particles for different states and substances. Their models have little explanatory power but they could still score on the measure of the macroscopic/collective dimension. About a third of the teachers had higher scores on the macroscopic/ collective dimension than the continuous/particulate dimension.

Overall, the pre-intervention correlations give some indication of a relationship between the nature of a person's particle model and its contribution to understanding physical phenomena.

Stronger indications have emerged in an analogous study with pupils, which used interviews to probe thinking (Author #1 & #3, 2005). Post-intervention correlations are much lower but this is probably a function of the much larger numbers of high scores all round.

To further explore any relationship between the particle model dimensions and understanding physical phenomena, lowest and highest achievement groups of teachers for each dimension were selected for a two-way ANOVA. Each lowest-achievement group includes every case with achievement score lower than M-3SE, and each highest-achievement group includes every case with achievement score higher than M+3SE, where M is the mean achievement and SE is the standard error of score distribution. For the continuous/particulate dimension the lower and higher groups numbered 73 and 62 respectively. The parallel numbers for the macroscopic/collective dimension are 83 and 54. A two-way ANOVA was carried out using the total achievement score on physical phenomena as the dependent The above provides further support to the suggestion that understanding physical phenomena is related to understanding the particulate theory of substances. The relationship is only apparent in the pre-intervention measures, which reflect residual knowledge left from the teachers' formal education. This association is not too surprising. Those holding a weak particle model are not likely to find it useful for explanations. The key issue is the direction of any causality. Does particle theory open up the understanding of physical phenomena or vice versa? Our data do not provide any evidence either way. The large effect of the intervention obscures any relationship in the post-intervention scores.

Which teachers benefited from the course?

To give the overall picture, Figure 2 shows the distributions of the teachers' total scores for the pre and post tests. The effectiveness of the intervention per task is shown in Table 9. For each task the difference between pre-test/ post-test mean scores is statistically significant (p < 0.0001). In addition, the effect sizes (Type error II) estimated by using Pearson's r-vales [START_REF] Field | Meta-analysis of correlation coefficients: a Monte Carlo comparison of fixed and random effects methods[END_REF][START_REF] Rosenthal | Contrasts and effect sizes in behavioural research: a correlational approach[END_REF] indicate that the magnitudes of these changes were substantial. Overall, the effect size on the total score is 0.89. Nonparametric tests, such as Wilcoxon Signed-Ranks test led to the same conclusions.

[Insert Figure 2 about here] and [Insert Table 9 about here] The Spearman's rho correlation coefficient between pre-intervention and postintervention total test scores is 0.05. Previous understanding is not a good predictor of success. Figure 3 [Insert Figure 3 about here]

Effect of gender and teaching experience

Table 10 compares the test means for males and females. For both pre and post intervention there are no significant gender differences. To test the effect of teaching experience, lowest and highest experience groups were selected. The lowest-experienced group (LExp) includes all cases with teaching experience lower than M-3SE, and the highestexperienced group (Hexp) includes all cases with teaching experience higher than M+3SE, where M is the mean teaching experience and SE is the standard error.

[Insert Table 10 about here] Comparing the test means for these two groups, table 10 shows a significant effect of teaching experience on the post-intervention test. Even though the lowest-experienced group (LExp) and highest experienced group (HExp) scored equivalently in the pre-intervention test, post-intervention, the HExp group scored significantly lower (p < 0.05). The most experienced/ oldest teachers showed less progress, that is, the resistance to conceptual change was greater. This may indicate that less experienced teachers' ideas are less consolidated and more easily changed [START_REF] Sakonidis | Experts' and novices' primary teachers' intervening in students' mathematical activity: an insight[END_REF] or reflect more general differences between attitude to change and age (Hewstone & Stroebe, 2001). However, although statistically significant, the difference between the experience groups is small in comparison to the overall progress made by both groups.

Conclusions

The pre-intervention test showed that the primary teachers did not have a secure understanding of the physical phenomena and appeared to hold misconceptions similar to those that would be expected of their pupils. It seems reasonable to suppose that their thinking dates back to when they, themselves, were pupils. Furthermore, their experience of teaching these topics does not seem to have engendered any significant conceptual changes. There is nothing in the literature to suggest these teachers are atypical. A vicious cycle appears to be operating which allows misconceptions to persist from generation to generation. In an attempt to break this cycle, we have used a new conceptual approach to introducing the particle model in the context of physical changes. In this approach particle ideas are introduced within a substance-based framework rather than a 'solids, liquids and gases' framework. The focus is on explaining why a substance can be in any of the three states. Overall, the progress made by the teachers was encouraging. In comparison to in-service courses adopting constructivist teaching methodology within a 'solids, liquids and gases' approach we cannot say whether progress is better or worse. However, we would argue that our intervention has addressed important ideas, which teachers can take to and which offer the chance of breaking the cycle.

The almost zero correlation between the pre and post scores is in keeping with the course providing a new conceptual approach. As might be expected, the more experienced teachers were found to be more resistant to change but the effect was not large. Many of the older teachers did make good progress. However, good progress is not complete progress. The teachers made the better progress with melting and boiling. For these changes of state, the taught model considered the particle energy collectively without reference to energy distribution. Melting was explained by the particles having enough energy to partially overcome the hold between particles.

Boiling was explained by the particles having enough energy to completely overcome the hold. For a beaker of boiling water, the emphasis was on the water being in the gas state (the bubbles). The idea that enough water particles have enough energy (i.e. drawing on ideas of energy distribution) was not addressed. However, for evaporation below boiling point, ideas of energy distribution were introduced as a development of the particle model. This proved to be more difficult. Many teachers did not adopt ideas of energy distribution to explain room temperature evaporation. Similar difficulties have transpired in our work with primary pupils (Author#3 & #1). However, a 'reduced' model where room temperature evaporation is explained by bombarding air particles 'knocking out' water particles (without reference to energy) had more appeal. The parallel, here, is with dissolving rather than a change of state. This does make a distinction from boiling and perhaps is sufficient for primary level.

Identifying the condensation on a cold object as water was generally not a problem (pre and post). This implies appreciation that water exists within the air and this may be related to the teachers' willingness to accept the gas state. However, for most, the mechanism for condensation proved challenging. Understanding how water can exist as part of a gaseous mixture at room temperature and how this behaves on cooling is not easy. In addition to energy distribution, the idea of a balance between competing processes is also required: the chances of lower energy water particles meeting up versus the chances of clusters being broken up by higher energy particles (most likely air). After one intervention it is too early to judge whether such ideas are beyond most primary teachers and whether explaining the condensation of atmospheric water should be the preserve of specialist science teachers in secondary schools. The appropriateness for the primary curriculum is very doubtful. Notwithstanding issues surrounding mixtures of air and water, at the core of our approach is the use of the particle model to explain substances and their states. Our findings suggest this is accessible to primary pupils and their teachers. Like others (e.g. [START_REF] Jarvis | Changes in primary teachers' science knowledge and understanding during a two year in-service programme[END_REF] we would recommend continuous in-service training programs where ideas could be revisited. Such programs should start as soon as teachers enter the classroom. There seems to be no advantage in waiting for teachers to gain some classroom experience.

Furthermore, the present intervention course was focused on prerequisite content knowledge and attention should move on to enhancing pedagogical content knowledge (Boz & Boz, 2008;[START_REF] Grossman | The making of a teacher: Teacher knowledge and teacher education[END_REF][START_REF] Park | Revisiting the Conceptualisation of Pedagogical Content Knowledge (PCK): PCK as a Conceptual Tool to Understand Teachers as Professionals[END_REF]. A frequent reinforcement and development of the relevant ideas holds the potential for better teaching. Perhaps, in time, it might be possible to tip the vicious cycle into a virtuous cycle. Table 1. Outline of content of the teaching scheme concerning physical changes. 1. Properties and the material/object distinction Some properties depend on the material only. Some properties depend on the material, the amount (dimensions of an object) and the shape/structure of an object.

Definition of a substance.

Melting behaviour can be used to distinguish between a pure sample of a substance and a mixture of substances.

A simple particle model

Particle ideas can explain melting. A sample of a substance was presented as a collection of particles with empty space between. Key points were:

• The particles have an ability to 'hold on' to each other:

• They are always moving in some way (energy of movement): and • The particles of a particular substance remain the same in a change of state.

A sample of a substance could be in one of three states.

A sample of a substance can be in the gas state. Explanations for the phenomena of melting and boiling Why different substances can be in different states at room temperature.

Mixing and unmixing.

Distribution of energy among the particles of a substance. Evaporation below boiling point into the air. Condensation of atmospheric water vapour. 1. Teachers were asked to draw a relevant picture and to explain what they draw: There were some pictures given in order to stimulate their imagination (Figure 1). Teachers could pick up one of these pictures or draw their own, in case none of these satisfied them. In any case teachers should explain what the picture presents indicating and naming any part of the picture.

2. Teachers were asked to answer this question only if there were a reference to particles in their previous answers.

Otherwise, they encouraged to proceed to part II.

3. A macroscopic description of a piece of wax melting is given as stimulus. Teachers were asked to name the material after melting and to describe the structure of wax before and after melting drawing corresponding microscopic picture (teachers could also use pictures of Figure 1).

4. A macroscopic picture of boiling water inside a beaker is given as stimulus. Teachers were asked to name the material in the liquid area, inside a bubble and right upper the surface of boiling water and to describe the structure of these three areas drawing corresponding microscopic pictures (teachers could also use pictures of Figure 1).

5.

A macroscopic description of evaporation (below boiling point) of water is given as stimulus. Teachers were asked to describe and explain the phenomenon of evaporation (of water), as well as, to make a distinction between boiling and evaporation.

6. A macroscopic description of the formation of drops on a cool surface of a can is given as stimulus. Teachers were asked to name the material of drops and give explanations for their formation.

  program. Under central control from the Greek Ministry of National Education and Religion Affairs each Primary Educational Department of Greek Universities had the responsibility for planning and implementing a particular training program. These were open to Greek primary teachers with up to 25 years teaching experience, on a voluntary basis. In this context, Democritus University of Thrace launched a two-year program, where, among other courses, participants attended a five week (six hours per week) training course on basic concepts of chemistry. The conceptual structure and content of the course had been developed by two of the authors (the 1 st and the 3 rd ).

  At the beginning of the course the teachers completed a pre-intervention written test consisting of two parts (Table2). Part I concerns the understanding of the particle model itself and Part II concerns the application of the model to explain phenomena. In sum, Parts I and II relate to the first research question. Setting the two parts against each other addresses the second research question.

  about here]

  of matter Pre-and post-intervention responses concerning the continuous/particulate dimension of substances (Table

  , appreciating that changes in motion and spacing alone (regardless of individual particle 'physical' nature) are sufficient to explain different states appears to be easier for a change of state than different substances in different states.

  here] Boiling Pre intervention, similarly to pupils' answers in other studies (e.g. Authors #1 & #3, 2005; Author #3, 1998b; Hatzinikita & Koulaidis, 1997; Osborne & Cosgrove, 1983; Paik, Page 12 of 39 URL: http://mc.manuscriptcentral.com/tsed Email: ijse_editor@hotmail.co.

  been quite ineffective with regard to understanding this common phenomenon. Postintervention, increases to 33% for the category C and 43% for PC indicate a positive effect of . However, it is quite clear that the challenge posed by evaporation at room temperature requires further attention.

  and macroscopic/collective dimensions separately, Table

  depicts the changes between pre and post course performance in more detail. The different fill patterns labeled A, B, C, D and E represent the 15 th , 35 th , 50 th , 65 th and 85 th percentiles of the post course scores respectively. With the pre-intervention scores divided into five bands, the shading patterns indicate the achievements of the teachers in each pre-course score band. All percentile destinations on the post test are found for each score band on the pre-test.

  .,Cakiroglu, J., & Ozkan, O. (2004). Turkish preservice science teachers' understanding of science and their confidence in teaching it. Journal of Education for Teaching, 30(1), 57-68. Tytler, R.(2000). A comparison of year 1 and year 6 students; conceptions of evaporation and condensation: Dimensions of conceptual progression. International Journal of ScienceEducation, 22(5), 447-467. Woolnough, B.(1994). Effective science teaching, Buckingham, Open University Press.

  )= Single particles of ice/water/vapor same -Differences specified in relevant distances and motions. PC (partially correct)= Single particles of ice/water/vapor are alike in shape (size) but have different size (shape). Differences also reported in relevant distances and/or motions (poor justification). I (incorrect)= macroscopic character on particles (or no answer). 5a,b C (correct)= single particles of different substances are different in size and shape (complete justification) -Differences also reported in relevant distances and motions. PC (partially correct)= single particles of different substances are different (poor justification or no justification). NEI (not entirely incorrect)= single particles of different substances are different (the difference in the state prevails), I (incorrect)= macroscopic character on particles (or no answer).

  correct)= same substance in liquid state. PC (partially correct)= not definitely same substance. I (incorrect)= different substance (or no answer). 6b C (correct)= correct structure for both solid (pre) and liquid (after melting) states. PC (partially correct)= correct structure of one state and not clearly specified structure of the other. NEI (not entirely incorrect)= not clearly specified structure for both states. I (incorrect)= incorrect structure for both states (or no answer). 7a C (correct)= water in gas state. PC (partially correct)= reference to a gas, not clearly water vapor. NEI (not entirely incorrect)= air. I (incorrect)= oxygen and or hydrogen (or no answer). 7b C (correct)= correct structure for both liquid and gas states. PC (partially correct)= correct structure of one state and not clearly specified structure of the other. NEI (not entirely incorrect)= not clearly specified structure for both states. I (incorrect)= incorrect structure for both states (or no answer).

Figure 1 :Figure 2 :

 12 Figure 1: Some of the pictures, which were included in the test as a stimulus. Clarifications on what the picture presents, are also given below the pictures. [Picture 1: A continuous colourless material, Picture 3: Small droplets, the one touch on the other, Picture 5: particles (like small spheres, the one does not touch on the other, in certain positions), Picture 8: particles (like small spheres, the one away from the other, in random positions)] 224x69mm (96 x 96 DPI)

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	F o r
	P
	e
	e r
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	w
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	y

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  variable with Continuous/Particulate and Macroscopic/Collective as independent variables.Results are shown in Table8for the pre-intervention scores. Levene's test does not reject the equality of variances (p=0.84), thus accuracy in the final statistical test is expected. The two-

	way ANOVA for pre-intervention test [F (3,108)=30.4, p < 0.000] explained 44.3% (Adj. R2
	0.44) of the total variance: The main effect of the Particulate dimension is statistically
	significant and it explained 36.2% of the variance [F (1,108)=61.1, p < 0.000]. The main
	F effect of Collective dimension is statistically significant but very small -it explained 4.1% of o r the variance [F (1, 108)=4.57, p < 0.035]. Interaction effects of Particulate x Collective were
	not statistically significant. P e [Insert Table 8 about here]
	e r
	R
	e
	v i e
	w
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	y

Table 2 .

 2 Description of the instrument. Parts and tasks.

	Parts	Description of Parts	Tasks	Description of Tasks
	I	Particulate-Continuous	Solid state	1	Description of what can be seen inside a grain of sugar, if it could be magnified a huge amount. 1
			Liquid state	2	Description of what can be seen inside a drop of water, if it could be magnified a huge amount. 1
					Description of what can be seen inside a spotted area of
			Gas state	3	oxygen (in gas state in a vase), if it could be magnified a
					huge amount. 1
		Macroscopic-Collective	Same Substance in different states	4	Description and comparison between single particles of water in the three states. 2
			Different substances in different states at room temp.	5	Description and comparison between single particles of sugar, water and a gas in room temperature. 2
	II	Physical Phenomena	Melting	6	Description and comparison between the two states of wax during melting, i.e., before and after melting. 3
					Description and comparison among three areas of
			Boiling	7	boiling water, i.e., the liquid area, inside a bubble and
					upper the surface of boiling water. 4
			Evaporation	8	Description /explanation of evaporation of water. 5
			Condensation	9	Description /explanation of condensation of water on a cool surface. 6

Table 3 .

 3 Categories of teachers' responses (n=162) in Continuous/Particulate dimension. Preand post-intervention frequencies* for substances in the three states of matter at room temperature.

	Category	Score		Solid	Liquid	Gas
			Pre	Post	Pre	Post	Pre	Post
	I	0	48	6	37	0	40	1
	NEI	1	82	24	67	9	21	14
	PC	2	25	75	43	77	74	64
	C	3	7	67	15	76	27	83
	C (correct)= complete picture of microscopic structure.
	PC (partially correct)= notion of particles but not clearly address the nature of the free space.
	NEI (not entirely incorrect)= particles in continuous matter.
	I (incorrect)= continuous matter, irrelevant or no answer

Table 4 .

 4 Categories of teachers' responses in Macroscopic-Collective dimension. Pre-and post-intervention frequencies* (n=162).

	Task	Category Score	Frequencies
				Pre intervention	Post intervention
	4a-Comparison	I	0	75	16
	between particles in different states	PC	1	43	28
	(Same substance)	C	2	44	118
	4b-Description of	I	0	118	34
	particles in different states	PC	1	17	25
	(Same substance)	C	2	27	103
	5a-Comparison	I	0	100	76
	between particles in different states	NEI	1	17	10
	(Different substances)	PC	2	10	16
		C	3	35	60
	5b-Description of	I	0	114	62
	particles in different states	NEI	1	11	49
	(Different substances)	PC	2	37	32
		C	3	0	19

Table 5 .

 5 Categories of teachers' descriptions concerning melting wax and boiling of water (n=162). Pre-and post-intervention frequencies.

	Task	Category	Score	Frequencies
				Pre intervention	Post intervention
	6a-Identity of the	I	0	20	0
	melted wax	PC	1	12	1
		C	2	130	161
	6b-Structure of	I	0	32	3
	the substance before and after	NEI	1	38	3
	melting	PC	2	64	10
		C	3	28	146
	7a-The content	I	0	42	2
	of the bubbles	NEI	1	63	24
		PC	2	12	26
		C	3	45	110
	7b -Structure of	I	0	57	7
	liquid and gas states	NEI	1	40	6
		PC	2	22	16


MD Katarina Stingl
email: katarina.stingl@med.uni-tuebingen.de

Esther Hoffmann

Ulrich Schiefer

Adaptation of a digital Camera for Simultaneous Stereophotography in Ophthalmology

Keywords: digital simultaneous stereophotography, glaucoma follow-up documentation, optic nerve disc photography, fundus camera

Simultaneous stereoscopic fundus photography is an important tool for classification and follow-up of glaucomatous optic neuropathy. The use of conventional film-based simultaneous stereoscopic fundus cameras is complicated and time-consuming due to film processing and sensitivity of its mechanical components. Digital simultaneous stereoscopic fundus cameras are not available in Germany, since the existing ones are lacking the EC-certificate. We realised a digital simultaneous stereophotographic fundus camera by replacing the conventional film-based analogue camera of the simultaneous stereoscopic fundus camera TOPCON TRC-SS (TOPCON CORPORATION, Tokyo, Japan) by the digital 21.10 megapixel, 36 x 24 mm full frame CMOS camera CANON EOS 5D, Mark II (CANON INC., Tokyo, Japan).

BACKGROUND

In clinical ophthalmology morphological findings are crucial for diagnostics, as well as follow up or accessing therapeutic success. Of particular importance is a stereoscopic view of the posterior eye segment structures such as the retinal morphology, the optic nerve head, or the retinal vessels. A monocular estimation is not appropriate for a three dimensional estimation. [START_REF] Martinello | 3-D retinal surface inference: stereo or monocular fundus camera?[END_REF]. A stereo image is regarded the standard for assessing optic disc damage in glaucoma. [START_REF] Pablo | Optic nerve head changes in early glaucoma: a comparison between stereophotography and Heidelberg retina tomography[END_REF][START_REF] Hoffmann | Optic disc photography and retinal nerve fiber layer photography[END_REF]. The three dimensional morphologic finding can be accessed by the examiner with the help of binocular indirect ophthalmoscopy and the slit lamp; however, documentation for classification and follow-up purposes requires a stereoscopic picture.

An exact and reproducible three dimensional documentation is necessary for clinical routine follow up as well as for a documentation in clinical studies. [START_REF] Budenz | Detection and prognostic significance of optic disc hemorrhages during the Ocular Hypertension Treatment Study[END_REF][START_REF] Miglior | Results of the European Glaucoma Prevention Study[END_REF][START_REF] Gaasterland | The Advanced Glaucoma Intervention Study (AGIS): 10. Variability among academic glaucoma subspecialists in assessing optic disc notching[END_REF]. To our knowledge, a simultaneous stereoscopic fundus camera capable of digital imaging, such as NIDEK 3-Dx (NIDEK Co., Ltd., Hiroishi, Japan) is not approved in Germany due to lacking CE-certification. According to the outing of the distributors of the OCULUS Company (OCULUS, Wetzlar, Germany) the light emission of this camera is assumed to be too high, interfering thus with the certificate. Sequential stereoscopic imaging by shifting the monocular fundus camera has proven to be insufficient due to inappropriate standardization. [START_REF] Krohn | Comparison of photographic techniques and films used in stereophotogrammetry of the optic disk[END_REF][START_REF] Chang | Diagnosing glaucoma progression[END_REF]. The aim of this paper is to demonstrate a solution for upgrading a conventional film-based simultaneous stereoscopic camera into digital equipment.

METHODS

The conventional simultaneous stereo fundus camera TOPCON TRC-SS (TOPCON CORPORATION, Tokyo, Japan) has been transformed into a digital camera by replacing the original camera body by a digital camera (CANON EOS 5D, Mark II, CANON INC., Tokyo, Japan), as depicted in Fig. 1. The 21.10 megapixel full frame CMOS (complementary metal oxide semiconductor) sensor camera is capable of capturing both stereo-images simultaneously. The connection of the digital camera was provided by a specifically constructed adapter connecting the conventional fundus camera and the digital full format camera.

RESULTS AND DISCUSSION

To our knowledge, this is the first time that a conventional analogue simultaneous stereoscopic fundus camera has been transformed into a digital system, which is compatible with the CE prescription and can be integrated into an electronic patient data record system. Stereoscopic fundus photography is an optimal documentation of the morphology of the posterior eye segment in follow up of glaucoma or macular diseases. [START_REF] Budenz | Detection and prognostic significance of optic disc hemorrhages during the Ocular Hypertension Treatment Study[END_REF][START_REF] Miglior | Results of the European Glaucoma Prevention Study[END_REF][START_REF] Gaasterland | The Advanced Glaucoma Intervention Study (AGIS): 10. Variability among academic glaucoma subspecialists in assessing optic disc notching[END_REF]9,[START_REF] Davis | The Age-Related Eye Disease Study severity scale for age-related macular degeneration: AREDS Report No. 17[END_REF]. Such findings can be collected and viewed by stereo viewers such as the screen-VU Stereoscope (Heidelberg Engineering, Inc., Heidelberg, Germany). The result of conventional stereoscopic photography approaches are classical photo slides. This approach is, although the picture quality is mostly excellent, time-consuming, since the photography film must be used up completely before development, which is usually performed externally in case of colour slides. As a consequence, an immediate observation and judgement of the photographic output is impossible. Moreover, the storage and retrieval of photographic slides is a considerable economical and spatial burden. Switching to digital photography allows circumventing the above mentioned shortcomings and is extremely attractive for the increasing number of clinics working with digital patient records as is the case of the Department and Institute for Ophthalmology, University of Tuebingen. For gaining electronic data, the classical analogue stereoscopic slides can be digitalized ex post. However, this is a considerable economical and time effort that is, unfortunately, associated with an inevitable quality loss. Therefore, an optimal solution, direct stereoscopic digital fundus photography should be obtained.

A simultaneous stereoscopic fundus camera with the possibility of digital imaging, such as NIDEK 3-Dx (NIDEK Co., Ltd., Hiroishi, Japan) has not been approved in Germany so far. The reason for this is the lacking CE-certification, since the light emission of this camera is assumed to be too high, based on the information of the distributors of the OCULUS Company (OCULUS, Wetzlar, Germany). Alternatively, pseudo-stereoscopic images with a standard digital fundus camera can be obtained. In this case, two nonstereoscopic pictures are taken subsequently from two different angles mimicking thus a stereo view.

Unfortunately, a pseudo-stereoscopic picture is not an optimal solution for a lack of reproducibility as the amount of shift is depending on the (varying) pupil diameter and on the subjective approach of the photographer. For example, an inconsistency of the camera angle can lead to false positive or false negative judgment with regard to progression in glaucoma. [START_REF] Chang | Diagnosing glaucoma progression[END_REF][START_REF] Knudsen | A 3-dimensional evaluation of the macular region: comparing digitized and film-based media with a clinical evaluation[END_REF]. Therefore, the ideal method for stereoscopic documentation is simultaneous stereophotography, whereas pseudo-stereoscopic techniques are less frequently used nowadays.

Recent follow-up imaging techniques such as the Heidelberg Retina Tomograph (HRT, Heidelberg Engineering, Inc., Heidelberg, Germany) have shown to be comparably accurate for diagnosing the optic nerve head damage. [START_REF] Pablo | Optic nerve head changes in early glaucoma: a comparison between stereophotography and Heidelberg retina tomography[END_REF], however, in estimating glaucoma progression criteria such as haemorrhages or disc pallor cannot it is inferior to stereophotography. So far, a good stereophotography of the optic nerve head remains the best method for the morphological evaluation in glaucoma.

Examples of a stereoscopic optic disc finding documentation are shown in Fig. 2 and Fig. 3. For viewing a classical stereo-viewer or software for quantitative analysis can be applied.

Since analogue simultaneous stereoscopic fundus cameras such as TOPCON TRC-SS were broadly used in the earlier decades and may be part of the equipment of many ophthalmologic departments and clinics, we present a simple and a low-cost solution for converting this high-quality documentation system into a digital one in order to document the stereoscopic image. An important aspect is that the attached digital camera must have a full frame sensor (36 x 24 mm) in order to capture the full image produced by the conventional TOPCON fundus camera, which would not be possible with cameras with smaller sensors.

CONCLUSIONS

As a conclusion, by transferring a conventional photo-based simultaneous stereoscopic camera into a digital unit we were able to realize a solution for 3D morphological documentation in ophthalmology without the disadvantages of the analogue stereoscopic photography (financial and time effort as well as spacy storage, retrieval requirements and non-suitability for direct transfer into electronic records) as well as those of the pseudo-stereophotography (lack of reproducibility).

FIGURE LEGENDS

Fig. 1 :

 1 Fig. 1: The analogue simultaneous stereo fundus camera TOPCON TRC-SS has been transformed into a digital system by adding the digital camera CANON EOS 5D, Mark II (21.10 megapixel, 36 x 24 mm full frame CMOS sensor).

Fig. 2 :

 2 Fig. 2: A simultaneous digital stereoscopic picture of an optic nerve head obtained with the introduced digital simultaneous stereophotography fundus camera. The pictures can be viewed directly on the computer screen by means of a stereo viewer (e.g. Screen-VU Stereoscope, Heidelberg Engineering, Inc., Heidelberg, Germany). Normal optic disc.

Fig. 3 :

 3 Fig.3: A simultaneous digital stereoscopic picture of an optic nerve head obtained with the introduced digital simultaneous stereophotography fundus camera. The pictures can be viewed directly on the computer screen by means of a stereo viewer (e.g. Screen-VU Stereoscope, Heidelberg Engineering, Inc., Heidelberg, Germany). Glaucomatous optic disc.

ACKNOWLEDGEMENT

This project was supported by ALCON Pharma GmbH, Freiburg, Germany.

COMPETING INTERESTS

None

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in British Journal of Ophthalmology and any other BMJPGL products to exploit all subsidiary rights, as set out in the licence.


MD Alex Hørby Christensen
email: alexhc@dadlnet.dk

MD Marianne Benn

PhD Henning Bundgaard

MD, DMSc Anne Tybjaerg-Hansen

MD, DMSc Stig Haunso

MD, DMSc Jesper Hastrup Svendsen

Wide Spectrum of Desmosomal Mutations in Danish Arrhythmogenic Right Ventricular Cardiomyopathy Patients

Keywords: ARVC, Genetics, Desmosome, Cardiomyopathy

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a lethal condition characterized by ventricular tachyarrhythmias, right-and/or left ventricular involvement and fibrofatty infiltrations in the myocardium. The disease has been associated with mutations in genes encoding desmosomal proteins.

Objective: To thoroughly evaluating an ARVC cohort for desmosomal mutations and large genomic rearrangements and characterized the phenotype associated with double mutation carrier status.

Methods: Sixty-five unrelated patients (55 fulfilling current criteria and 10 borderline cases) were screened for mutations in all known desmosome genes (DSC2, DSG2, DSP, JUP, PKP2) and TGFb3. Presence of genomic rearrangements was assessed by multiplex ligation-dependent probe amplification.

Results:

The screening identified 19 different mutations: two mutations in DSG2, four in DSC2, two in DSP (one heterozygous; one homozygous), four in JUP (one patient compound heterozygous) and seven in PKP2. No genomic rearrangements or mutations in TGFb3 were identified. Ten of the mutations were novel. Seven families carried more than one mutation. Clinical evaluation of these families showed a variable phenotype associated with the double mutation carrier status. The homozygous desmoplakin mutation (DSP p.G2056R+p.G2056R) carrier came from a consanguious Danish family and had left ventricular involvement, palmar keratoderma and curly hair consistent with a Carvajal-like syndrome.

Conclusions: Thirty-three percent of patients in this Danish ARVC cohort carried desmosomal mutations with a surprisingly wide mutation spectrum. A substantial proportion of patients carried more than one mutation. Our study supports comprehensive desmosomal mutation screening beyond the first encountered mutation, while routine screening for genomic rearrangements does not seem indicated.

Introduction

Arrhythomogenic right ventricular cardiomyopathy/dysplasia (ARVC; MIM# 107970) is an inherited cardiomyopathy characterized by ventricular arrhythmias, sudden cardiac death, right and/or left ventricular involvement, and fibrofatty infiltrations in the myocardium. A clinical diagnosis is made according to published Task Force criteria. 1 2 The original criteria [START_REF] Mckenna | Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy. Task Force of the Working Group Myocardial and Pericardial Disease of the European Society of Cardiology and of the Scientific Council on Cardiomyopathies of the International Society and Federation of Cardiology[END_REF] were published in 1994, followed by modified, more sensitive, criteria for first degree relatives in 2002. [START_REF] Hamid | Prospective evaluation of relatives for familial arrhythmogenic right ventricular cardiomyopathy/dysplasia reveals a need to broaden diagnostic criteria[END_REF] Recently, revised Task Force criteria were published [START_REF] Marcus | Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: Proposed Modification of the Task Force Criteria[END_REF] and these include a number of changes: quantification of histology and right ventricular size, novel electrocardiographic features, genetic testing, and several other parameters are now part of the diagnostic criteria.

Seven genes have so far been associated with the disease. Five of these genes encode desmosomal proteins: desmocollin-2 (DSC2; MIM# 610476) 4 5 , desmoglein-2 (DSG2; MIM# 610193) [START_REF] Pilichou | Mutations in desmoglein-2 gene are associated with arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Syrris | Desmoglein-2 mutations in arrhythmogenic right ventricular cardiomyopathy: a genotypephenotype characterization of familial disease[END_REF][START_REF] Bhuiyan | Desmoglein-2 and Desmocollin-2 Mutations in Dutch Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Patients: Results from a Multicenter Study[END_REF][START_REF] Awad | DSG2 mutations contribute to arrhythmogenic right ventricular dysplasia/cardiomyopathy[END_REF] , desmoplakin (DSP; MIM# 607450) 10 11 , plakoglobin (JUP; MIM# 611528) 12 13 , and plakophilin-2 (PKP2; MIM# 609040) [START_REF] Gerull | Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Van Tintelen | Plakophilin-2 mutations are the major determinant of familial arrhythmogenic right ventricular dysplasia/cardiomyopathy[END_REF][START_REF] Christensen | Missense Variants in Plakophilin-2 in Arrhythmogenic Right Ventricular Cardiomyopathy Patients -Disease-Causing or Innocent Bystanders?[END_REF] . The non-desmosomal gene, transforming growth factor beta-3 (TGFb3; MIM# 107970) [START_REF] Beffagna | Regulatory mutations in transforming growth factorbeta3 gene cause arrhythmogenic right ventricular cardiomyopathy type 1[END_REF] , has also been suggested to be involved in the pathogenesis of ARVC. Recently, a gene of unknown function, TMEM43 (MIM# 604400), was found causative in ARVC5. [START_REF] Merner | Arrhythmogenic right ventricular cardiomyopathy type 5 is a fully penetrant, lethal arrhythmic disorder caused by a missense mutation in the TMEM43 gene[END_REF] The most common form of inheritance is autosomal dominant with variable penetrance and phenotypic expression. In general, the dominantly inherited forms have an isolated cardiac phenotype while the recessive forms also have variable degrees of hair and skin abnormalities.

Homozygous mutations in DSP [START_REF] Alcalai | A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia, skin disorder, and woolly hair[END_REF][START_REF] Uzumcu | Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome[END_REF][START_REF] Norgett | Recessive mutation in desmoplakin disrupts desmoplakinintermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma[END_REF] and JUP [START_REF] Mckoy | Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease)[END_REF] have been reported in two cardiocutaneus syndromes named Carvajal Syndrome (MIM# 605676) and Naxos disease (MIM# 601214), respectively. This study is an extension of our previous work investigating the prevalence of PKP2 mutations in Denmark. [START_REF] Christensen | Missense Variants in Plakophilin-2 in Arrhythmogenic Right Ventricular Cardiomyopathy Patients -Disease-Causing or Innocent Bystanders?[END_REF] First, we aimed systematically at screening all known desmosome genes without excluding known mutation carriers from further analysis. Secondly, we aimed at clinically evaluating family members in families carrying more than one mutation. And finally, we wished to evaluate the contribution of genomic rearrangements to the pathogenesis of ARVC.

Methods

Study population

The study population comprised a total of 65 unrelated, white individuals. Fifty-five (32 men) patients fulfilled ARVC 1994 Task Force criteria and ten patients (six men) had some ARVC characteristics but did not fulfil the criteria. All patients were identified at a tertiary referral center, Rigshospitalet, Copenhagen University Hospital. The patients were diagnosed in the period 1993-2008. The clinical characteristics of all index patients are summarized in table 1. Initial clinical evaluation of family members routinely consisted of physical examination, 12-lead ECG, signalaveraged ECG, transthoracic echocardiography, 48-hour Holter monitoring, and MRI.

The study was approved by the local ethics committee (KF01-152/04), followed the Helsinki Declaration II, and all participants gave written informed consent prior to inclusion.

Genetic analyses

For all living patients genomic DNA was extracted from whole blood using standard techniques.

For deceased patients DNA was extracted from paraffin-embedded tissue (individual III:1, Family G and the proband, Family J) or Guthrie card (individual III:4, Family A). Polymerase chain reaction (PCR) amplification of all exons and flanking introns of the DSC2, DSG2, DSP, JUP, PKP2, and TGFb3 genes was performed using standard protocols and PCR fragments were bidirectionally sequenced on an ABI Prism 3730 Analyzer (Applied Biosystems, USA). PCR primers are listed in supplementary table 1. Some fragments were pre-screened using Denaturing High Performance Liquid Chromatography (dHPLC) (Transgenomic, USA) or a LightScanner System (Idaho Technology, USA). Samples pre-screened with dHPLC were mixed with wild type PCR product to enable detection of homozygous variants. DNA extracted from tissue or Guthrie card was amplified and sequenced without any pre-screening. The following reference sequences were used: DSC2 (NM_024422/NM_004949), DSG2 (NM_001943), DSP (NM_004415), JUP (NM_002230), PKP2 (NM_004572) and TGFb3 (NM_003229).

Presence of large genomic insertions and deletions was evaluated with a Multiplex Ligation-dependent Probe Amplification (MLPA) Salsa P168 ARVC kit (MRC-Holland, the Netherlands). The kit has probes for DSP, JUP, PKP2, RYR2 and TGFb3. The analyses were performed in accordance with the supplier's protocol, which consisted of four steps: 1) denaturation of DNA and annealing of MLPA probes, 2) ligation, 3) multiplex PCR reaction with universal primers and 4) fragment analysis using capillary electrophoresis.

For all identified mutations at least 650 self-reported healthy, ethnically matched control individuals were screened using Taqman assays (Applied Biosystems, USA) or a LightScanner System.

A mutation was defined as a sequence variant absent in at least 1300 control chromosomes, and affecting one or more conserved nucleotides/residues, or introducing a change in amino acid polarity in a functionally important protein domain. All other sequence changes were referred to as variants. For clarity, only patients who carried at least one mutation were included in diagnostic yield percentages.

Statistical analysis

For comparisons of frequencies of relevant mutations in the patient and control population Fisher's exact two-sided probability tests were used. A p-value <0.05 was considered significant.

Results

Genetic findings

Eighteen different heterozygous and one homozygous mutation were identified. The mutations occurred in 18 patients fulfilling Task Force criteria. No mutations were found in the borderline group. The mutations identified (table 2) consisted of two in DSG2 (p.T335A, p.E1020AfsX18), four in DSC2 (p.E2K, p.G150A, p.S824L, c.2508+5G>A), two in DSP (c.939+1G>A, p.G2056R+p.G2056R), four in JUP (p.S225L, p.V407I, p.T427M, p.V603L) and seven in PKP2 (previously reported [START_REF] Christensen | Missense Variants in Plakophilin-2 in Arrhythmogenic Right Ventricular Cardiomyopathy Patients -Disease-Causing or Innocent Bystanders?[END_REF] ). No genomic rearrangements or mutations in TGFb3 were identified. Ten of the mutations were novel. All were absent in a minimum of 650 controls. Six rare variants (DSC2 p.A897KfsX4, DSG2 p.V56M, DSG2 p.V920G, DSP p.C81Y, PKP2 p.D26N, PKP2 p.S140F) occurred in both the patient and control populations. As these variants may contribute to the pathogenesis of ARVC they are mentioned where appropriate. Sequence alignments, in silico analysis, and complete listing of all identified non-synonymous variants are provided in supplementary table 2 and 3. Six families (Families A-F) were digenic mutation carriers, one family compound heterozygous for two plakoglobin mutations (Family G), one family had a homozygous desmoplakin mutation (Family H), and nine families (Families I-L and five families only carrying PKP2 mutations not presented) had a single mutation.

Desmocollin-2

Four mutations were identified in DSC2. The DSC2 p.E2K mutation affects the second residue in the signal peptide which is cleaved of before insertion into the plasma membrane. The patient carrying the DSC2 p.E2K mutation also carried the PKP2 p.S140F variant (Family D). Two other identified mutations, DSC2 p.G150A and DSC2 p.S824L, both affect highly conserved residues in functionally important parts of the protein. The DSC2 p.G150A mutation co-occurred with the JUP p.S225L (Family A) mutation and the DSC2 p.S824L mutation carrier (Family E) also had the PKP2 p.D26N variant. The DSC2 c.2508+5G>A splice site mutation (Family I) affects a highly conserved nucleotide in the splice donor site of intron 15. One proband and one borderline patient carried the reported DSC2 p.A897KfsX4 (c.2687_2688insGA) variant. However, this variant was also present in the control population (two out of 65 patients versus 22 out of 650 controls, p=1.0).

Desmoglein-2

Two mutations were identified in DSG2. One missense mutation (DSG2 p.T335A, Family J) affecting a highly conserved residue and one out-of-frame deletion (DSG2 p.E1020AfsX18, Family K). One proband and one borderline patient carried the reported DSG2 p.V56M (c.166G>A) and

Large genomic rearrangements

All patients had normal MLPA profiles excluding large genomic insertions and deletions in the DSP, JUP, PKP2, RYR2 and TGFb3 genes.

Clinical findings

The clinical features of the mutation carriers are summarized in table 3.

In family A (figure 1A), the proband was diagnosed with ARVC at age 18 years prior to her sudden death. The patient's first-degree relatives were evaluated without abnormal findings. Seven years later her 16 year old sister unfortunately died and autopsy showed fibrofatty infiltrations in the myocardium. All family members were re-evaluated and the mother and father, an uncle and two aunts fulfilled criteria for ARVC. Genetic screening revealed a truncating PKP2 mutation (PKP2 p.S329EfsX24) on the maternal side and two different missense mutations on the paternal side (JUP p.S225L and DSC2 p.G150A). All mutations individually segregated with disease. Individual III:4 carried both the fathers and mothers' mutations. Individual II:8, who had a different digenic carrier status (DSC2 p.G150A and JUP p.S225L), had significantly left ventricular involvement (ejection fraction of 30%). Her son (individual III:6) carried the same two mutations, but had a positive signal-averaged ECG as the only abnormal diagnostic finding at the age of 33 years. An ECG, microscopy photo and an electropherogram from the family are shown in figure 2A-C.

In Family B (figure 1B), the proband had symptom onset at the age of 24 years and was diagnosed with ARVC. His father and brother also had features of ARVC. All family members had normal left ventricular function and there was no history of sudden cardiac death. At the time of diagnosis the proband had documented ventricular tachyarrhythmias, but for the past 11 years of follow-up he has not received ICD-therapy. His father (individual I:1), currently aged 75 years, has not had any ARVC-related symptoms for 20 years. Genetic screening identified two mutations (PKP2 p.R79X and JUP p.S225L). The proband and his father were digenic carriers and the brother only carried the PKP2 p.R79X mutation.

In Family C (figure 1C) the proband had syncopes and ventricular tachycardia and was diagnosed with ARVC. Family evaluation showed that his younger brother (individual II:3) fulfilled Task Force Criteria. His older brother (individual II:4) also had some ARVC characteristics. Both the probands daughters (individual III:1 and III:2) were phenotypically normal. Genetic screening identified one splice site mutation (PKP2 c.2146-2A>T) and one rare variant (DSP p.C81Y) in the proband, but only the PKP2 mutation in the clinically affected younger brother. One of the daughters carried the DSP p.C81Y variant.

In Families D, E and F (figures 1D-F) all evaluated family members were phenotypically normal and carried none of the probands mutations.

In Family G (figure 1G) the proband died suddenly aged 31 years and autopsy revealed fibrofatty infiltrations in the myocardium. Clinical evaluation of the mildly symptomatic mother showed premature ventricular contractions, but otherwise normal clinical findings. Her brother and son (individuals II:3 and III:2) were asymptomatic but had some diagnostic features of ARVC.

Genetic screening showed that the proband was JUP p.T427M mutation carrier, the mother was compound plakoglobin mutation carrier (JUP p.T427M and JUP p.V603L) and the mother's brother and her sons were JUP p.V603L mutation carriers. The two mutations segregated with disease individually.

In Family H (figure 3A-D) the proband had symptom onset at the age of 35 years and was diagnosed with ARVC with left ventricular involvement. In addition, palmar keratoderma was noted (figure 3C). No significant plantar skin manifestations or woolly hair were seen. His mother died driving a moped aged 35 years and his father died of cancer aged 78 years. The mother and father were cousins. None of the probands three children had any cardiac symptoms. The proband carried a homozygous DSP p.G2056R mutation and his parents and children were obligate heterozygous mutation carriers. His brother and sister declined clinical and genetic evaluation.

Discussion

On the basis of our present results combined with our previous PKP2 screening we conclude that 33% of patients in this Danish ARVC cohort had mutations in desmosomal genes. Our cohort had a surprisingly wide mutation spectrum with a high frequency of JUP and DSC2 mutations. Previous studies have shown 10-43% PKP2 mutations 14-16 22 , 0-12% DSG2 mutations 6-9 23 24 , 1-16 % DSP mutations 6 11 24 and 0-5 % DSC2 mutations 4 8 23 24 . Only three dominant mutations in JUP have previously been described. 13 24 25 A single report has associated mutations in TGFb3 with ARVC1 [START_REF] Beffagna | Regulatory mutations in transforming growth factorbeta3 gene cause arrhythmogenic right ventricular cardiomyopathy type 1[END_REF] , but mutations have not been described in other populations. However, most previous studies have excluded patients after the first identified mutation.

In six families (11%) digenic mutation carriers were identified where at least one of the mutations were absent in the control population. In Families A, B and G the individual mutations segregated with the disease and were individually regarded as pathogenic. In family A the digenic mutation carriers had a severe phenotype with sudden cardiac death and left ventricular involvement. In contrast, the digenic carriers in family B and G had a mild phenotype with minimal arrhythmia, no left ventricular involvement or sudden cardiac death. Two recent papers suggested a gene dosage effect with patients carrying more than one mutation having a more severe phenotype. 8 26 Our data partially support these findings although the phenotypic variation is large. In Family C the disease segregated with one of the mutations (PKP2 c.2146-2A>T) suggesting that the rare variant (DSP p.C81Y) is not necessary for the development of the ARVC phenotype. Whether the identified rare variants also present in our control population are benign or have a diseasemodifying effect is unknown. In other inherited cardiac arrhythmia syndromes polymorphisms can modify the phenotype. [START_REF] Poelzing | SCN5A polymorphism restores trafficking of a Brugada syndrome mutation on a separate gene[END_REF] One patient from a consanguinous Danish family (Family H) was a homozygous DSP mutation carrier. Homozygous mutations have been reported for all five desmosomal genes. Except for the homozygous DSG2 and PKP2 mutation carriers all have had variable degrees of hair and skin abnormalities. A single report has shown a homozygous DSP missense mutation (p.G2375R+p.G2375R), located in the B domain of the C-terminal part of the protein, as cause of Carvajal Syndrome. [START_REF] Alcalai | A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia, skin disorder, and woolly hair[END_REF] The reported case had significant hair-and skin involvement with woolly hair and a pemphigous-like skin disorder. Interestingly, our patient with a similar glycine to arginine substitution (p.G2056R+p.G2056R) in a homolog region located in the A domain, only showed mild cutaneous abnormalities and no woolly hair. The C-terminal part of desmoplakin consists of three plakin repeat domains each composed of four and a half copy of a unique 38 amino acid repeat motif. Functional evidence suggests that all three domains have similar binding properties to the intermediate filaments. [START_REF] Choi | Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure[END_REF] The variable phenotype between the two homozygous missense mutation carriers is therefore not likely to be caused by a weaker binding of the intermediate filaments to the A domain. Our patient had significant left ventricular involvement consistent with two out of three reports on homozygous DSP mutation carriers. [START_REF] Alcalai | A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia, skin disorder, and woolly hair[END_REF][START_REF] Uzumcu | Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome[END_REF][START_REF] Norgett | Recessive mutation in desmoplakin disrupts desmoplakinintermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma[END_REF] In addition to homozygous mutations, compound heterozygous and dominant DSP mutations with variable degrees of cardiac and cutaneous involvement have been reported. [START_REF] Asimaki | Unique epidermolytic bullous dermatosis with associated lethal cardiomyopathy related to novel desmoplakin mutations[END_REF] The present study furthermore aimed at investigating large genomic rearrangements. Only a single report from the Netherlands has implicated large genomic deletions in the pathogenesis of ARVC with a diagnostic yield below one percent. [START_REF] Van Der Smagt | Large Genomic Deletions in Plakophilin-2 are a Rare Cause of ARVD/C and ARVD/C-like Disease[END_REF] However, only 26 patients out of the 120 patients screened in this study fulfilled Task Force criteria. [START_REF] Van Der Smagt | Large Genomic Deletions in Plakophilin-2 are a Rare Cause of ARVD/C and ARVD/C-like Disease[END_REF] Our results are in keeping with the Dutch study, as no rearrangements were identified in our cohort. Although a substantial part of the reported desmosomal mutations are truncating with haploinsufficiency as the proposed molecular mechanism, large genomic deletions does not seem to be a common cause of ARVC. In contrast to this, MLPA analysis of a selected group of long QT syndrome patients showed that 11.5% of patients had disease-causing large genomic rearrangements. [START_REF] Eddy | Identification of large gene deletions and duplications in KCNQ1 and KCNH2 in patients with long QT syndrome[END_REF] None of the 10 borderline patients had potentially disease-causing mutations as only two variants of unknown significance (DSC2 p.A897KfsX4 and DSG2 p.V920G) were found. This finding may be coincidental and reflect the small sample size. The average period of follow-up was long in these patients (data not shown) suggesting that time-dependent reduced expressivity is not the case, and that these patients may represent phenocopies.

The following limitations apply to this study: cohort size, referral bias to tertiary center, a limited number of family members available for evaluation, issues related to mutation analysis on DNA extracted from paraffin-embedded tissue, and the lack of functional tests available for verification of the disease causing effect of identified mutations. Furthermore, the MLPA kit had a limited number of probes and the sensitivity of the kit may not be 100%. Due to few patients carrying more than one mutation and unclear pathogeneity of a number of the identified mutations no meaningful statistical genotype-phenotype comparisons between single and double mutation carriers were possible. Lastly, due to the anonymous nature of our control population no systematic evaluation for ARVC was possible and it cannot be definitively ruled out that some controls had silent disease.

In conclusion, 33% of the patients in this Danish ARVC cohort had desmosomal mutations with a wide mutation spectrum and a substantial proportion of patients carried more than one mutation. The phenotype associated with the double carrier status was variable. Our data clearly support comprehensive mutation screening beyond the first encountered mutation, while routine screening for large genomic rearrangements does not seem indicated.

  DSG2 p.V920G (c.2759T>G) variants, respectively. These variants were also present in the control population (1/65 vs. 4/650, p=0.38; 1/65 vs. 3/650, p=0.32). Desmoplakin Two mutations were identified in DSP. The DSP c.939+1G>A mutation (Family L) disrupts the conserved splice donor site of intron 7 and the homozygous DSP p.G2056R mutation (Family H) affects the highly conserved domain A of the C-terminal part of the protein. One patient (Family C) carried the DSP p.C81Y (c.242G>A) variant. This patient also carried the PKP2 c.2146-2A>T splice site mutation. The DSP p.C81Y variant was present in two out of the 1400 controls screened for this variant (1/65 vs. 2/1400, p=0.13) Plakoglobin Five mutations in JUP were identified in four patients. All mutations affect highly conserved residues in the armadillo repeat domains. The JUP p.S225L mutation occurred in two unrelated patients, of which one came from a family (Family A) with two other mutations resulting in two different digenic carrier statuses within the same family (JUP p.S225L+PKP2 p.S329EfsX24 and JUP p.S225L+DSC2 p.G150A, respectively). The other JUP p.S225L mutation occurred in a family (Family B) also carrying the PKP2 p.R79X mutation. The mother of a sudden cardiac death victim carried two plakoglobin mutations (JUP p.T427M and JUP p.V603L, Family G). The JUP p.V407I mutation carrier (Family F) also had the PKP2 p.D26N variant. Transforming growth factor beta-3 No mutations were found in the coding sequence, flanking introns, 5' untranslated region or 500 base pairs upstream of the TGFb3 gene. Two novel polymorphisms (c.*739C>T and c.*831G>A) were found in the 3' untranslated region.

Figure 1 .

 1 Figure 1. Pedigrees of families with digenic or compound inheritance. Women are illustrated by

Figure 2 .

 2 Figure 2. A) ECG from individual II:8 (Family A), who had significant left ventricular involvement,

Figure 3 .

 3 Figure 3. A) Pedigree of the family (Family H) carrying the homozygous DSP p.G2056R mutation.

Table 1 . Clinical characteristics of the cohort according to the 1994 Task Force criteria.

 1

	ARVC group	Borderline group

Table 2 . Overview of the mutations.

 2

	Family	Gene	Nucleotide change	Amino acid or RNA change	Change in polarity	Evolutionary conservation	Novel	Protein domain
	A, III:4	PKP2	c.983_984insGG	p.S329EfsX24 *			-	N-terminal
		JUP	c.674C>T	p.S225L	+	+	+	Arm3
	A, III:6	DSC2	c.449G>C	p.G150A	-	+	+	EC1
		JUP	c.674C>T	p.S225L	+	+	+	Arm3
	B	JUP	c.674C>T	p.S225L	+	+	+	Arm3
		PKP2	c.235C>T	p.R79X			-	N-terminal
	C	PKP2	c.2146-2A>T	r.spl			-	Arm8
		DSP	c.242G>A	p.C81Y	-	+	+	N-terminal
	D	DSC2	c.4G>A	p.E2K	-	-	+	Signal peptide
		PKP2	c.419C>T	p.S140F	+	-	-	N-terminal
	E	DSC2	c.2471C>T	p.S824L	+	+	+	Cytoplasmic
		PKP2	c.76G>A	p.D26N	-	+	-	N-terminal
	F	JUP	c.1219G>A	p.V407I	-	+	+	Arm7
		PKP2	c.76G>A	p.D26N	-	+	-	N-terminal
	G	JUP	c.1280C>T	p.T427M	+	+	+	Arm8
		JUP	c.1807G>T	p.V603L	-	+	+	Arm11
	H	DSP	c.6166G>C+c.6166G>C	p.G2056R+p.G2056R	+	+	+	DACT
	I	DSC2	c.2508+5G>A	r.spl?			+	Cytoplasmic
	J	DSG2	c.1003A>G	p.T335A	+	+	-	EC3
	K	DSG2	c.3059_3062delAGAG	p.E1020AfsX18			+	Cytoplasmic
	L	DSP	c.939+1G>A	r.spl			-	N-terminal

Arm: Armadillo repeat; DACT: Domain A, C-terminal; EC: Extracellular repeat. Variants present in control population are in shaded grey. * Reported as PKP2 E329fsX352 due to alternative nomenclature.

Acknowledgements

We thank all participating patients and their families. We also thank Claus B. Andersen for providing the photomicrograph.

Funding This work was supported by grants from The Danish National Arrhythmia Research Foundation Centre for Cardiac Arrhythmia (DARC), Danish Cardiovascular Research Academy (DaCRA), The Research Council at the Heart Centre, Rigshospitalet, The Villadsen Family Foundation, Brødrene Hartmanns Foundation, and The John and Birthe Meyer Foundation.

Competing interests

None.

Patient consent

Obtained.