Roberto Sarmiento
email: sarmientoroberto@yahoo.com.mx

Vinaya Shukla
email: shuklav1@cf.ac.uk

Zero-sum and frontier trade-offs: an investigation on compromises and compatibilities amongst manufacturing capabilities

Keywords: MANUFACTURING STRATEGY, REGRESSION ANALYSIS, BENCHMARKING, HEURISTICS, STATISTICAL trade-off model, compatibilities, cumulative capabilities, zero-sum trade-off model, compatibilities, cumulative capabilities, zero-sum trade-offs, frontier trade-offs, theory of performance frontiers, probabilistic analyses, deterministic analyses, top performers/extreme cases, thought experiments

Zero-sum and frontier trade-offs: an investigation on compromises and compatibilities amongst manufacturing capabilities

Introduction

The importance that trade-offs amongst an organisation's objectives have on its strategy (and hence its competitiveness) has been commented on and discussed by prominent authors in both the business (e.g.; [START_REF] Porter | Competitive Strategy: Techniques for Analyzing Industries and Competitors[END_REF][START_REF] Porter | Competitive Advantage: Creating and Sustaining Superior Performance[END_REF][START_REF] Mintzberg | The Strategy Process: Concepts, contexts, cases[END_REF] and manufacturing (e.g.; [START_REF] Skinner | Manufacturing-Missing Link in Corporate Strategy[END_REF][START_REF] Skinner | The Focused Factory[END_REF][START_REF] Hayes | Restoring our competitive edge: Competing through manufacturing[END_REF][START_REF] Hill | Manufacturing strategy: Text and cases[END_REF] strategy fields. The operations management authors in particular have commented that a successful strategy is one that is built around strategic manufacturing trade-offs. Structural (e.g.; production capacity and technology, facilities) and infrastructural (e.g.; quality management systems, human resources management, information technology systems) resources have to be deployed and developed accordingly. Moreover, it is argued that the failure to recognise the existence of these trade-offs could result in a loss of competitive power at the industry/marketplace level [START_REF] Hayes | Restoring our competitive edge: Competing through manufacturing[END_REF].

A salient theme from this line of research is the approaches and rationales by which researchers, especially in manufacturing strategy studies, have examined and come to the conclusion that different capabilities (e.g.; quality, delivery, cost) are traded-off or compatible. This is a particularly important issue in investigations that employ quantitative approaches to the analysis of the information (e.g.; linear regression/correlation analysis).

In this paper, we intend to address some of the limitations that past empirical and theoretical research on trade-offs and compatibilities between manufacturing capabilities has left pending. By means of a detailed analysis of well-accepted methodologies and assumptions, we arrive at the conclusion that, in order to resolve those issues, two types of compromised relationships between different capabilities need to be recognised: "zero-sum" and "frontier" trade-offs. Zero-sum trade-offs are commonly represented by a statistically significant and negative relationship between two capabilities. Generally speaking, this kind of trade-off is one in which a gain in performance in one capability entails a loss in performance in another capability in a more direct/linear manner. On the other hand, the frontier type of trade-off is characterised by a differential in performance between two capabilities after some level of compatibility is achieved, something that is consistent with the concepts and ideas advanced by various authors (e.g.; [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF]Schmenner and Swink,1998). It is noted that frontier trade-offs are difficult (if not impossible) to identify with probabilistic and overall associative approaches to the analysis of a 2 sample of subjects (e.g.; linear regression/correlation analysis, path analysis). Thus, it is proposed that additional and more focused and deterministic (i.e.; precise) analyses, including those with a heuristic "benchmark" approach also be employed. It can also be said that zero-sum trade-offs represent a relationship between two capabilities that can be far more "adversarial" than when two capabilities are in a frontier trade-off. As such, the differential in performance caused by a zero-sum compromise is expected to be considerably greater than the one caused by a frontier trade-off.

We discuss the findings of our investigation in light of past theoretical and empirical research. For example, we argue that when some studies have claimed to offer evidence against the existence of trade-offs between different capabilities in their samples, they are really offering evidence only against the existence of zerosum trade-offs, but not against the potential existence of frontier trade-offs. Zerosum compromises might be contingent upon various circumstances and conditions (e.g.; [START_REF] Safizadeh | Revisiting alternative theoretical paradigms in manufacturing strategy[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF]. Also, when authors have acknowledged and/or provided evidence that some trade-offs can be improved but not eliminated (e.g.; [START_REF] New | World-class manufacturing versus strategic trade-offs[END_REF][START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF][START_REF] Da Silveira | Exploring the trade-off concept[END_REF], they have arguably made an implicit reference to a frontier trade-off that governs the relationships between different criteria. The potential presence of frontier trade-offs implies a change in the way compromises and compatibilities amongst manufacturing capabilities are understood. This means that, as opposed to what current rationales propose, compatibilities (e.g.; cumulative capabilities) and (frontier) trade-offs can coexist within the same sample of subjects. To the best of our knowledge, this is the first investigation that examines the issues outlined above. The consequences of these and other results are discussed, and proposals for future research are offered.

The rest of the paper is organised as follows: section 2 deals with the definitions and background of our investigation. Specific research questions are laid out. Section 3 presents an analysis on well-accepted approaches and rationales, and compares them with more recent theoretical developments. Inconsistencies and limitations are highlighted, and the existence of zero-sum and frontier trade-offs is advanced in order to resolve some of those issues. Section 4 compares the findings of our investigation with those of previous research, and section 5 offers conclusions and suggestions for future studies.

Definitions

In order to achieve clarity, several concepts and definitions need to be established. Therefore, a trade-off situation is defined as one in which a firm achieves high levels of performance in one manufacturing capability at the expense of lower levels of performance in one or more capabilities (i.e.; a gap in performance that represents a differential in competitive power). This definition is consistent with the trade-off model's original tenets: "…. no technologically-based system can perform equally well on every performance criterion ….." (Skinner, 1996, p. 8). In this context, a "high" level of performance is defined as an "… outstanding enough …. to create competitive advantage" (Skinner, 1996, p. 6) level of achievement in one or more capabilities. The preceding statements imply that, at the very least, different capabilities that are traded-off will not attain an "outstanding enough to create competitive advantage" type of performance at the same time and under the same circumstances. This definition includes the meaning of the "frontier" and "zero-sum" trade-offs introduced in section 1.

A compatibility situation implies that there is evidence of an overall and similar degree of performance achievement up to a determined level between different capabilities. This definition of "compatibility" covers the characterisation of "cumulative capabilities" that has been used by previous authors (e.g.; [START_REF] Ferdows | Lasting improvements in manufacturing performance: In search of a new theory[END_REF][START_REF] Noble | Manufacturing strategy: Testing the cumulative model in a multiple country context[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF]. Nonetheless, contrary to the definition of "cumulative capabilities", "compatibility" does not necessarily imply, for We also advance a heuristic and deterministic (i.e.; precise) approach to analyse compromises and compatibilities between competitive criteria. The firms are first identified according to the levels of performance they achieve across the different capabilities included in the analysis. Then, for each individual firm, we make assessments of performance between different pairs of manufacturing capabilities in terms of the mutual levels of achievements and also the shortfalls/differentials in performance with respect to higher levels of achievement. This, in turn, will allow us to focus on the "top performers" (i.e.; a benchmark approach) in order to understand more fully the trade-offs and/or compatibilities that might exist between different pairs of capabilities. The "top performer (s)" is defined as the firm (s) that observes the highest levels of achievement across multiple capabilities in a sample. This would mean that, for instance, the ideal top performer (s) would be the firm (s) that achieves an outstanding enough to create competitive advantage type of performance across all manufacturing capabilities at the same time. Section 3 explains this approach in more detail. Schmenner and Swink (1998, p. 108) affirm that an asset frontier is defined by and dependent upon the kinds of investments that appear on the fixed asset portion of the balance sheet. Furthermore, they assert that, as firms approach their asset frontier, trade-offs between different capabilities will become more evident. These relationships are characterised by the laws of diminishing returns and synergy 1 .

Figure 1 illustrates these concepts:

________________

Take in figure 1 _________________

Theoretical background

There has been much interest in examining whether manufacturing firms can become industrial/marketplace leaders across several or all manufacturing capabilities. The underlying idea, evidently, is to achieve excellence in performance that in turn will provide firms with a competitive advantage. Let us consider Skinner's actual words: "one system cannot be outstanding enough at meeting all criteria to create competitive advantage" (Skinner, 1996, p. 6). Also: "… no technologicallybased system can perform equally well on every performance criterion" (Skinner, 1996, p. 8). Putting those two statements together, the conclusion is that the trade-off model actually predicts that no manufacturing system can perform equally well and outstandingly enough across every performance criterion at the same time.

Therefore, it is recommended that companies should adopt a "focused factory" approach to the design, development, and implementation of the strategy and operations of a production unit. Echoing Skinner's arguments, Hayes and Wheelwright (1984, p. 41) also warn against trying to compete by offering superior performance in all manufacturing capabilities, since any attempt to do so would likely result in firms achieving only a second-best type of performance in all of them, something that in turn would make them lose competitiveness. It follows from the preceding discussion that in order to conclusively assert that no trade-off is present in a relationship between any pair of capabilities, a firm or firms would have to attain an "outstanding enough to create competitive advantage" type of performance across those capabilities at the marketplace/industrial level of analysis and under the same circumstances.

On the other hand, various authors have proposed what they consider to be competing, more complete and comprehensive models and theories. For example, the cumulative capabilities models (e.g.; [START_REF] Ferdows | Lasting improvements in manufacturing performance: In search of a new theory[END_REF]) and rigidflexibility models (e.g.; [START_REF] Collins | An empirical test of the rigid flexibility model[END_REF] offer alternative views on the development of strong capabilities. The basic idea behind these models is that some (or all) tradeoffs can be overcome, particularly if continuous improvement programs such as TQM, JIT, TPM, etc., are successfully implemented. It is also worth noting that it is not clear whether the proponents of the cumulative capabilities and rigid-flexibility models affirm that all trade-offs can be overcome or simply that a production unit can be "outstanding enough to create competitive advantage" on more than one or two capabilities. A literature review identifies a number of studies that deal with diverse aspects of the trade-off model and/or its competing concepts, with varying results and conclusions [START_REF] Ferdows | Lasting improvements in manufacturing performance: In search of a new theory[END_REF]Roth andMiller, 1990, 1992;[START_REF] Noble | Manufacturing strategy: Testing the cumulative model in a multiple country context[END_REF][START_REF] Schroeder | Manufacturing performance trade-offs: An empirical investigation[END_REF][START_REF] Mapes | Performance trade-offs in manufacturing plants[END_REF][START_REF] Morita | The linkage amongst management systems, practices and behaviour in successful manufacturing strategy[END_REF][START_REF] Szwejczewski | Delivery and trade-offs[END_REF]Fillipini et al, 1998;[START_REF] Collins | An empirical test of the rigid flexibility model[END_REF][START_REF] Flynn | World class manufacturing: An investigation of Hayes and Wheelwright's foundation[END_REF][START_REF] Fynes | Quality practices, quality performance and business performance: A test of the "trade-off" and "multiple capability" theories[END_REF][START_REF] Mapes | Process variability and its effect on plant performance[END_REF][START_REF] Safizadeh | Revisiting alternative theoretical paradigms in manufacturing strategy[END_REF][START_REF] Dostaler | Trying to resolve manufacturing performance trade-offs: The case of British electronic contract electronic assemblers[END_REF][START_REF] Dostaler | Beyond practices: A qualitative inquiry into high performance electronics assembly[END_REF] da Silveira [START_REF] Da Silveira | Exploring the trade-off concept[END_REF][START_REF] Corbett | Searching for the sand cone in the GMRG data[END_REF][START_REF] Corbett | Key manufacturing capability elements and business performance[END_REF][START_REF] Stratton | The strategic integration of agile and lean supply chain[END_REF][START_REF] Lapre | Performance improvement paths in the U. S. airline industry: Linking trade-offs to asset frontiers[END_REF][START_REF] Rosenzweig | Towards a theory of competitive progression: Evidence from high-tech manufacturing[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF][START_REF] Narasimhan | An exploratory study of manufacturing practice and performance interrelationships[END_REF][START_REF] Fynes | The impact of supply chain relationship dynamics on manufacturing performance[END_REF][START_REF] Da Silveira | Improving trade-offs in manufacturing: Methods and illustration[END_REF][START_REF] Da Silveira | Effects of simplicity and discipline on operational flexibility: An empirical re-examination of the rigid flexibility model[END_REF][START_REF] Grobler | An empirical model of relationships between manufacturing capabilities[END_REF][START_REF] Squire | The impact of mass customisation on manufacturing trade-offs[END_REF][START_REF] Swink | Faster, better, cheaper: a study of NPD project efficiency and performance tradeoffs[END_REF]Stratton and Warburton, 2006;[START_REF] Sarmiento | Delivery reliability, manufacturing capabilities and new models of manufacturing efficiency[END_REF][START_REF] Antonio | The impact of product modularity on competitive capabilities and performance: An empirical study[END_REF][START_REF] Sarmiento | Manufacturing capabilities and performance: a critical analysis and review[END_REF]).

An analysis of the aforementioned studies shows some evidence that firms are capable of achieving synergies, mutual progression, and in some cases, what could be considered as high levels of performance on a number of capabilities.

Nonetheless, it can also be concluded that the main tenets of the trade-off model have not been refuted. In other words, the reviewed investigations do not offer evidence supporting the existence of a firm or firms that have actually achieved an "outstanding enough to create competitive advantage" level of performance across all manufacturing capabilities.

A key topic in this area of research concerns the way in which authors have determined that two or more capabilities are traded-off or compatible. The reviewed studies show a favoured approach to the examination of these themes: researchers obtain from firms cross-sectional data (perceptual and/or objective) representing manufacturing performance metrics (e.g.; cost, delivery). The data is then analysed using quantitative and probabilistic approaches (e.g.; linear regression/correlation analysis and related methodologies). Generally, analysts have come to the conclusion that different capabilities are in a trade-off or in a compatibility (e.g.;

"cumulative capability") situation if they observe significant negative or positive correlation coefficients, respectively. For example, Safizadeh et al (2000, p. 117) write: ".. a positive correlation means parallel developments of two capabilities. A negative correlation implies trading-off one capability for another". Such definitions imply that, since the trade-off model and its seemingly competing theories have been depicted as mutually exclusive concepts, previous authors have chosen to assert the presence of two competing concepts (e.g.; trade-offs or cumulative capabilities)

based on mutually exclusive types of evidence (i.e.; significant negative or positive correlations) that describe overall associations amongst variables within a sample of subjects. The type of compromised relationship described in those studies is consistent with the zero-sum trade-off introduced in section 1. As previously mentioned, the above methodologies and rationales have been widely accepted and used in past research. Nevertheless, we think that more recent theoretical developments have important repercussions on those approaches. For example, various authors in the operations and strategic management areas (e.g.; [START_REF] New | World-class manufacturing versus strategic trade-offs[END_REF][START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF][START_REF] Schmenner | On theory in operations management[END_REF][START_REF] Porter | What is strategy?[END_REF] have suggested that while there may be instances in which different criteria can observe mutual improvements and parallel trajectories along their development, strategic trade-offs and compromises are still existent, relevant, and should not be overlooked.

Thus, if compatibilities and compromises can be observed along the development of two capabilities, it follows that approaches capable of identifying both trade-offs and compatibilities in a relationship are necessary. Such approaches should be consistent when the unit of analysis is a group of firms or individual firms. These premises suggest a reassessment and re-evaluation of current methodologies and rationales used in these types of studies in order to determine whether they are consistent with newer theoretical developments. Consequently, when contradictions or inconsistencies are found, they need to be resolved.

The re-evaluation of well-accepted methodologies and rationales raises a number of important questions. For example, are regression/correlation analyses capable of identifying the potential trade-offs and compatibilities that can exist between a pair of competitive criteria as implied by more recent theoretical investigations? If not, what types of approaches are needed? If new approaches are needed, how can they be reconciled and made consistent with past methods and rationales? The rest of the paper will attempt to answer these questions.

An investigation on trade-offs and compatibilities

Consistent with Schmenner and Swink (1998), we utilise "thought experiments" to illustrate and support our arguments. The use of thought experiments is a well-accepted approach in areas such as physics [START_REF] Popper | The logic of scientific discovery[END_REF].

Nevertheless, some researchers (e.g.; [START_REF] Folger | Theorising as the thickness of thin abstraction[END_REF] have also discussed the usefulness of such an approach in business management studies. Therefore, our use of this type of experiment is justified. By means of a detailed analysis of a framework that is representative of more recent theoretical developments, we explore the potential advantages and limitations of widely accepted methodologies and rationales. To resolve some of the issues that past investigations did not identify and

Rationale for the existence of frontier trade-offs

To support our arguments for the existence of frontier trade-offs, we use the illustration advanced by Schmenner and Swink (1998, figure 1 in this paper).

However, one addition is made in order to highlight some issues within that framework. Figure 2 shows this:

____________

Take in figure 2 ____________

We have added a positive regression/correlation line to Schmenner and Swink's (1998) original framework to represent the "cumulative capabilities" they say are present in their illustration. As was discussed in section 2, a positive and significant linear regression/correlation coefficient is a widely used and well-accepted means to depict "cumulative capabilities". Furthermore, figure 2 suggests a positive and significant trajectory, as the firms included in it are portrayed to move upwards and to the right along the development of their capabilities. Therefore, we think that our inclusion of the regression/correlation line in figure 1 is valid. Having said this, we also identify a particular issue: Schmenner and Swink (1998) argue for the existence of both "cumulative capabilities" and "trade-offs" in their framework. They also comment that such compromises are observed after cumulative capabilities are attained, and that those trade-offs become more evident the closer a firm gets to its asset frontier. Thus, it can be said within reason that the trade-off implied by the theory of performance frontiers cannot be one that is consistent with an overall linear and negative regression/correlation analysis. As was discussed in section 2, such a coefficient is also a well-accepted means to assert the presence of a compromised relationship. To sum up, figure 2 implies a scenario in which both compatibilities and a trade-off that is not representative of a negative and significant regression/correlation line have to be fitted into one single framework.

To understand better and to attempt to resolve the above issues, we propose to extend the framework presented in figures 1 and 2 to include several firms at Since the study is illustrative in principle, the firms are modelled to have only two manufacturing capabilities (e.g.; quality, cost). We call these variables "MC1" and "MC2". The different performance levels in the firms are represented by a likert-type scale. These types of scales are a widely-used means to perceptually assess the relative performance of a firm compared to its industry/competitors 2 . This approach makes it consistent with the main tenets of the trade-off model that were discussed in section 2. The scale includes varying levels of performance: 5, 4, 3, 2, 1. In order to maintain consistency with [START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF], a "5" represents an "outstanding enough to create competitive advantage" level of performance, while the rest of the scales represent lesser levels of achievement. he/she would very likely conclude that "cumulative capabilities" exist between the two variables in the study, and that there is no evidence of any trade-offs. This would be based on the positive and significant regression/correlation line and coefficient obtained. Nevertheless, it is clear that such a conclusion would be incomplete. While

2 Although using perceptual measures of performance is consistent with the trade-off model's aims and goals, it could also be possible to utilise objective measures of achievement (e.g.; "98% on-time delivery rates, "30% of defective product"), as long as the sample includes firms from the same industry and similar context, or comparable competitors so that reasonable comparisons with respect to the marketplace-leading performers can be made. it is true that the positive and significant regression/correlation line would be an acceptable and overall indicator of the "cumulative capabilities" that exist in the framework (e.g.; a 1/1 ratio between MC2 and MC1 as shown by firms A, B, C and D), this method would not be able to identify the existing differential in performance between MC1 and MC2 (i.e.; a frontier trade-off) as observed in firm "E" (e.g.; a 4/5 ratio between MC2 and MC1). Such a compromise would be the result of the presence of the asset frontier as proposed by [START_REF] Schmenner | On theory in operations management[END_REF], which in turn would mean that only one capability would attain a level 5 in performance, while the other one would achieve only a lesser level of performance (e.g.; level "4"). This differential in achievement clearly would be in accord with the definition of trade-offs advanced by [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF]. The problem, nevertheless, resides in the fact that an overall methodology such as linear regression/correlation analysis would "mask" or "hide" the (frontier) trade-off caused by the asset frontier and that is expected to take place at the higher levels of performance. Arguably, methodologies based on the central tendency behaviour of the observations, such as linear regression/correlation analysis and related methodologies, would be incapable of detecting and distinguishing the differential in performance that is predicted to occur at the highest levels of the performance continuum and the closer a firm gets to its asset frontier.

Given these limitations, we think that a more correct and holistic analysis of the sample, as well as of individual observations, needs to include but also go beyond regression/correlation analysis. We propose a deterministic and heuristic approach to analyse each individual firm. Assessments with respect to both commonalities in performance achievements (e.g.; cumulative capabilities) and also shortfalls and differentials in performance (e.g.; trade-offs) amongst the criteria in discussion can be made. For example, let us re-examine figure 3, assuming that the researcher is not aware of the presence of a (frontier) trade-off. By analysing the firms individually, it could be said within reason that there is evidence of mutual progression and compatibilities in achievement between MC1 and MC2 up to the "4" level of performance, and that this trend is supported by an overall positive and significant regression/correlation coefficient. Nonetheless, by means of a deterministic "benchmark" analysis of the sample that focuses on the top performers (firms "D" and "E"), it can also be affirmed that the similar level of mutual progression, achievements, and compatibilities was not replicated at the level "5" of performance (i.e.; there is a differential in performance between MC1 and MC2, as seen in firm E), which in turn could be indicative of the presence of a (frontier) trade-off between MC1 . Arguably, focusing on the "best performers" (e.g.; firms that achieve "high levels of performance" in one or more capabilities) gives the researcher the best chance at analysing whether such high levels of performance could have come at the expense of lower levels of achievement in one or more different capabilities. Ideally, more focused and corroborative case studies (including longitudinal ones) would also be performed.

We think that our proposed heuristic approach and rationale to the analysis of the results is more complete than the way in which previous authors have examined the evidence and derived their conclusions in studies using a quantitative approach with cross-sectional data. For instance, various studies claim to present no results that support the existence of trade-offs in their samples 4 (e.g.; Ferdows and de [START_REF] Ferdows | Lasting improvements in manufacturing performance: In search of a new theory[END_REF][START_REF] Noble | Manufacturing strategy: Testing the cumulative model in a multiple country context[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF]. However, such a rationale is limited, because, while the lack of a significant and negative relationship might indicate the absence of a (zero-sum) trade-off, it is possible that a frontier trade-off could still be present, as our analyses have shown. To sum up, achievements in performance should be interpreted both in terms of the mutual levels of progression and compatibilities that the criteria under study observe, and also in terms of the performance shortfalls identified with respect to higher levels of achievement, which in turn could be indicative of the presence of a frontier trade-off.

We now proceed to justify the existence of zero-sum trade-offs.

Rationale for the existence of zero-sum trade-offs

It could be said that trade-offs of the zero-sum type, while not identified and labelled as such, is the most widely accepted form in which a compromised relationship between variables has been portrayed and understood. Our analyses of the trade-off model literature reveal that such an assumption is commonly used by previous investigations when assessing whether trade-offs between two different criteria exist, particularly in quantitative studies (e.g.; [START_REF] Ferdows | Lasting improvements in manufacturing performance: In search of a new theory[END_REF][START_REF] Safizadeh | Revisiting alternative theoretical paradigms in manufacturing strategy[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF]. Generally, a significant and negative regression/correlation coefficient has been taken as an indication of this type of 4 One way in which past studies assert the absence of a negative and significant relationship (and hence the absence of a (zero-sum) trade-off) is by means of a positive and significant coefficient between two capabilities. Another way is by means of a non-significant coefficient. ) observed particularly at the highest levels of performance. This is also consistent with the definition of a compromised relationship as proposed by [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF]. It is also evident that, as opposed to the case in which frontier trade-offs are present, linear regression/correlation analyses would be a fairly adequate tool to identify this latent trade-off.

The previous paragraphs have dealt with various issues that our research into the trade-off model and related concepts has raised. The acknowledgement of the existence of both zero-sum and frontier trade-offs reconciles some of those difficulties. This implies a change and re-evaluation of the rationales used to assert that two variables are in a compatible and/or compromised situation. Moreover, figures 3 and 4 show that the compromises in performance that zero-sum and frontier trade-offs represent are considerably different (e.g.; under a zero-sum trade-off, firm "E" would attain a "5" along MC3, but only a "1" in MC4; under a frontier trade-off, firm "E" achieves a "5" along MC1, but only a "4" in MC2). Also, it bears repeating that the presence of the asset frontier implies that both compatibilities (e.g.;

cumulative capabilities) and (frontier) trade-offs between two criteria can exist in the same sample of subjects, and that it is important to extend current methodologies and rationales used in these types of studies in order to acknowledge such a situation.

Discussion

Previous investigations offer some evidence of the several situations described and discussed in section 3. For example, two studies [START_REF] Safizadeh | Revisiting alternative theoretical paradigms in manufacturing strategy[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF] [START_REF] Da Silveira | Exploring the trade-off concept[END_REF] write that managers view trade-offs differently from academics, and that while some trade-offs can be improved, they cannot be eliminated. Their results

arguably could be indicative of the frontier trade-offs that we have identified. The consensus amongst researchers that have studied manufacturing trade-offs seems to be that evidence of a compromised relationship can only be asserted by means of a significant and negative correlation coefficient. Nevertheless, as our investigation has shown, a frontier type of trade-off is also a latent possibility that can explain why different capabilities, while observing mutual progression and compatibilities up to a certain level of performance, will not attain the same compatibility at higher levels of achievement.

We think that frontier trade-offs have a plausible theoretical basis. For example, [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF] and [START_REF] New | World-class manufacturing versus strategic trade-offs[END_REF] acknowledge the possibility that some trade-offs could be improved, but not eliminated, by the adoption of more advanced manufacturing technologies. [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF] in particular goes on to explain that two capabilities that are traded-off may run in parallel or counter to each other at different levels or amplitudes, but in the end, one will attain a higher level of performance than the other one. Thus, a pair of variables under the effect of a frontier trade-off could be indicative of Skinner's words: while they might be compatible even at the higher levels of performance, in the end, only one of them will attain an "outstanding enough to create competitive advantage" type of performance. [START_REF] Schmenner | On theory in operations management[END_REF] and their research into the asset frontier also offer strong support for the frontier trade-off concept identified in our investigation.

Another important issue concerns the potential causes of both zero-sum and frontier trade-offs. Various authors (e.g.; [START_REF] Schonberger | World class manufacturing: The lessons of simplicity applied[END_REF][START_REF] Schonberger | Building a chain of customers[END_REF][START_REF] Ferdows | Lasting improvements in manufacturing performance: In search of a new theory[END_REF][START_REF] Noble | Manufacturing strategy: Testing the cumulative model in a multiple country context[END_REF] imply that the absence of high implementation levels of JIT, TQM and related practices might cause a zero-sum trade-off between different criteria. Nevertheless, from the studies that present evidence of such trade-offs [START_REF] Safizadeh | Revisiting alternative theoretical paradigms in manufacturing strategy[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF], it can not be concluded that the negative and significant relationships observed are due to a lack of implementation of those practices. On the other hand, [START_REF] Safizadeh | Revisiting alternative theoretical paradigms in manufacturing strategy[END_REF] present an interesting result: zero-sum trade-offs seem to be dependent upon the production technology utilised. For example, "cost" and "delivery" observe a significant and negative relationship in batch shop processes, but no similar situation is reported for other types of production technologies. A similar scenario occurs between "quality" and "customisation", which has a zero-sum relationship in continuous flow shops, but no significant relationship in other types of production technologies. With regards to the causes of frontier trade-offs, from [START_REF] Schmenner | On theory in operations management[END_REF], we can derive that these compromises are caused by the asset frontier. Adding to the potential causes of competitive advantage, [START_REF] Vastag | The theory of performance frontiers[END_REF], in his comments on the theory of performance frontiers, argues that, excluding some special circumstances, the sources of sustained competitive advantage are more likely to come from infrastructural factors within firms (i.e. operating frontier) than from structural ones (i.e.; asset frontier). Also, [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF] suggests that structural and/or infrastructural factors might be at the root of trade-offs.

In terms of the compatibility situations depicted in our study (e.g.; significant and positive associations between two or more capabilities), these relationships are commonly found in previous studies (e.g.; [START_REF] Collins | An empirical test of the rigid flexibility model[END_REF]1998;Fynes et al;2000;[START_REF] Rosenzweig | Towards a theory of competitive progression: Evidence from high-tech manufacturing[END_REF][START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF]. These and similar investigations show a number of relationships in which different capabilities observe a positive and significant regression/correlation coefficient. Oftentimes, these compatibilities are associated with higher implementation levels of practices such as TQM, TPM and related programs (e.g.; [START_REF] Morita | The linkage amongst management systems, practices and behaviour in successful manufacturing strategy[END_REF][START_REF] Collins | An empirical test of the rigid flexibility model[END_REF][START_REF] Fynes | Quality practices, quality performance and business performance: A test of the "trade-off" and "multiple capability" theories[END_REF].

With regards to the more holistic approach discussed in sections 2 and 3, only two studies [START_REF] Filippini | Trade-off and compatibility between performance: Definitions and empirical evidence[END_REF][START_REF] Corbett | Key manufacturing capability elements and business performance[END_REF] were found to utilise a methodology similar to it. In those investigations, the authors group the subjects based on the levels of performance achieved along several capabilities and classify the firms in terms of the number of manufacturing capabilities in which they actually achieve what could be considered as a "high level of performance".

Interestingly, the results in both studies show that, generally, achieving high performance along multiple capabilities is difficult, a result that arguably is consistent with Skinner's trade-off and focused factory concepts (e.g.; 1969, 1974).

Furthermore, [START_REF] Filippini | Trade-off and compatibility between performance: Definitions and empirical evidence[END_REF], by means of a new methodology, establish the presence of trade-offs and compatibilities between some pairs of manufacturing capabilities, even when there is no significant correlation between them. For example, [START_REF] Filippini | Trade-off and compatibility between performance: Definitions and empirical evidence[END_REF] arrive at the conclusion that there are strong compatibilities between "delivery time" and "quality consistency", in spite of the absence of a significant and positive correlation between those capabilities. Like The preceding discussions have important implications. The use of probabilistic and linear regression/correlation analyses and related methods (e.g.;

path analysis) to determine the existence of trade-offs and compatibilities in a sample of subjects is a limited approach. A negative and significant regression/correlation coefficient might be a good indicator of the latent existence of a differential in performance between two variables, something that is consistent with the definition of a trade-off relationship. Nonetheless, as our investigation shows, in the case of significant and positive overall patterns, exclusive regression/correlation analyses are incapable of identifying the potential presence of a frontier trade-off. Such evidence between two capabilities might indicate a general level of compatibility. However, care should be exercised when interpreting such results, as a positive and significant regression/correlation line tends to "mask" or "hide" a frontier trade-off. Thus, a more holistic and deterministic analysis should acknowledge both the levels of compatibilities observed, and also the performance shortfalls with respect to higher levels of achievement, leaving open the possible presence of a frontier trade-off that would explain such shortfalls. Extra analyses focusing on the best performers (e.g.; a benchmark approach) are needed in order to identify the shortfalls in performance with respect to the highest levels of achievement.

It can also be concluded that, when past investigations (e.g.; Fynes et al;2000;[START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF] have claimed to find evidence that does not support the existence of trade-offs amongst the variables under study, in reality they have found evidence only against the zero-sum type of trade-off. The absence of a negative and significant relationship between two capabilities would seem to indicate that a gain in one would not necessarily mean a more direct, linear-type of loss in the other one. This would give basis to assert the absence of trade-offs of the zero-sum type. However, as we have shown, the absence of a zero-sum trade-off between two capabilities does not necessarily mean the absence of a frontier trade-off. Although these two trade-offs entail a differential in performance between two capabilities, they are considerably different, and require separate types of analyses in order to conclusively assert that they are not present within a sample. Consequently, future studies, while commenting on the absence of a significant and negative relationship between two criteria, could also make a distinction and conclude that evidence does different criteria can observe some degree of compatibility, a level after which a differential in performance will be observed. This is consistent with the performance frontier concept advanced by [START_REF] Schmenner | On theory in operations management[END_REF] and also with the research by other authors (e.g.; [START_REF] Skinner | Missing the links in manufacturing strategy[END_REF][START_REF] Skinner | Manufacturing strategy on the "S" curve[END_REF][START_REF] Porter | What is strategy?[END_REF]. Furthermore, both zero-sum and frontier trade-offs indicate that, generally speaking, different capabilities affected by either trade-off will not achieve an "outstanding enough to create competitive advantage" level of performance at the same time and under the same circumstances, something that is consistent with the foundational principles of the trade-off model. To the best of our knowledge, our study is the first one that formally establishes and recognises these different types of trade-offs and that also reconciles the differences between previous approaches and rationales and more recent theoretical advancements.

Frontier trade-offs imply a change in the way that compromised relationships are established in quantitative studies. It is important not only to analyse a sample with linear regression/correlation analyses. The possible presence of frontier tradeoffs also entails a need to rank the observations (e.g.; manufacturing firms) in terms of their actual performance achieved. This will allow the analyst to observe whether an "outstanding enough to create advantage" level of performance has been achieved along multiple capabilities by one or more firms, and also to determine if this phenomenon is consistent with overall trends within the sample as represented by significant correlation coefficients. Moreover, the absence of a negative and significant correlation coefficient between two variables has been interpreted as the absence of a compromised relationship. While this rationale can still be used to assert the absence of a zero-sum trade-off, unless there is conclusive evidence to the contrary, the potential existence of a frontier trade-off should be recognised. For example, when analysing multiple manufacturing capabilities, and in the presence of significant and positive correlations, it should be acknowledged that while there seems to be no evidence of zero-sum trade-offs, frontier trade-offs could still be present. Put differently, since the absence of zero-sum and frontier trade-offs can only be unmistakeably established if, and only if, different capabilities observe an "outstanding enough to create competitive advantage" level of performance at the same time, relationships in which at least one capability attains a lesser level of performance should be assessed considering the compatibilities and also the shortfalls in achievement observed, leaving the potential existence of frontier tradeoffs as a possible explanatory variable for the performance shortfalls.

The heuristic approach and rationale to the analysis of trade-offs and compatibilities that we are proposing can also be extended to research that includes more than two capabilities, both at the individual firm and at the group level. For example, let us suppose that a manufacturing firm observes 5, 5, 3, and 4 levels of achievement along "cost", "delivery", "quality" and "flexibility". Based on our rationale, it could be concluded that compatibilities amongst these capabilities are observed up to the "3" level of performance, a level after which varying performance achievements are observed, something that could be indicative of the existence of a frontier tradeoff amongst this group of capabilities. Frontier trade-offs can also be studied by means of longitudinal case studies with individual firms. As figure 3 and footnote 1 imply, a firm could observe a "5" and a "4" levels of achievement in various criteria.

Researchers could then investigate whether that gap in performance widens, disappears of stays unchanged over time, and the causes behind it.

Studies with real firms can not be expected to match the ideal conditions of our analytical investigation in section 3. From this premise, it follows that the issues raised by our research can become even more difficult in real-life studies. Therefore, we believe that our comments and proposals are even more relevant and valid when real-firms are included in an investigation. For example, the analyses and discussions in section 3 are based on the assumption of similar asset frontiers in all the participants. While this is useful to illustrate the nature of frontier trade-offs and its implications, it is possible that, in real-life investigations, a sample of subjects can include firms that have different asset frontiers, even when they belong to the same In such situations, our recommendation is to include as many manufacturing capabilities as possible in the analysis so that the shortfalls in performance with respect to higher levels of achievement can be identified. Since trade-offs are real and exist in all manufacturing firms, differentials in performance between pairs of competitive criteria should be expected and observed, either by means of regression/correlation analyses and/or other methodologies such as the benchmark approach we advance.

Although the questions posed at the end of section 2 have been answered already, we are laying them out again: linear regression/correlation analyses and related approaches are sufficient to identify overall compatibilities, but insufficient with regards to (frontier) trade-offs that can exist at the same time in a relationship between different criteria. New approaches need to be added to the analysis, including those with a "benchmark" focus as proposed in section 3. Past rationales and assumptions can be reconciled with more recent theoretical developments by acknowledging the existence of both zero-sum and frontier trade-offs.

Recently, [START_REF] Schonberger | Japanese production management: an evolution -with mixed success[END_REF] wrote that "… two decades later, trade-off viewpoints seem much suppressed. As [START_REF] Boyer | Competitive priorities: investigating the need for trade-offs in operations strategy[END_REF] suggest, trade-offs may be irrelevant in this hypercompetitive global economy that presses for improvement in multiple dimensions. Academic interest has shifted largely to the nature and subtleties of multiple or cumulative capabilities (e.g., [START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF])". We are modestly proposing that finer phenomena such as frontier trade-offs might be getting overlooked amongst overall positive and significant linear regression/correlation coefficients. While improvements and compatibilities might be achieved simultaneously along different criteria, we believe that trade-offs amongst them are still existent, relevant, and thus important for a long-lasting competitive advantage. We are inviting our colleagues to think of compromised relationships not only as those consistent with a zero-sum trade-off, but also to consider that frontier trade-offs are a real possibility. We hope that future researchers will take this up as a topic for their investigations.

  unresolved, we formally acknowledge the existence of zero-sum and frontier trade-offs.

  the development of their capabilities. Consistent with the assumptions made by[START_REF] Schmenner | On theory in operations management[END_REF], we also assume that the firms included in the study have similar asset frontiers and are comparable to each other.

Figure 3 3

 3 Figure33 illustrates the "cumulative capabilities" and (frontier) "trade-off" predicted by the theory of performance frontiers in an ideal form. It also includes different ways in which the sample of firms can be analysed.

  . As footnote 4 explains, the absence of such a coefficient has been explained as the lack of evidence of a compromised relationship between two variables. Figure4illustrates an ideal version of this type of trade-off: seen, the relationship shown above reflects a trade-off situation, a differential in performance between MC3 and MC4 (e.g.; fast delivery and marketbased quality, as in[START_REF] Flynn | An exploratory study of the nature of cumulative capabilities[END_REF]

  investigation also extends the definition of compatibilities and trade-offs beyond the obtention of significant positive and negative correlations.

  presence of trade-offs, but only of the zero-sum type, and that frontier trade-offs could still be present.5 Conclusions and suggestions for future researchThis investigation argues for the existence of two types of compromised relationships: Zero-sum and frontier trade-offs. These phenomena have been empirically and/or theoretically discussed and found in previous research, particularly in the operations management literature. Our proposal addresses some of the problems that past research has left unattended and that were discussed in section 3. Zero-sum trade-offs are characterised by a negative and significant relationship between different variables representing manufacturing capabilities. On the other hand, frontier trade-offs are hypothesised to be observed in a relationship in which

  and/or are direct competitors. In other words, different asset frontiers could result in frontier trade-offs between different pairs of criteria in different individual firms.

Figure 1 .

 1 Figure 1

1 The law of diminishing returns entails a proportional (e.g.; linear) relationship between two variables that becomes a disproportional (e.g.; nonlinear) one after a certain point in the relationship is reached. Thus, when Schmenner and Swink (1998) state that their framework rests on this premise, it can be derived plausibly that the "cumulative capabilities" they speak of in their framework are represented by the proportional aspect of the relationship, whereas "trade-offs" are exemplified by the non-proportional aspect of it (see for example the discussion in page 110 of Schmenner and Swink, 1998).

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  and scenarios (e.g.; production technology). Frontier trade-offs, on the other hand, have not been explicitly identified and discussed as such. Nevertheless, da Silveira

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have shown evidence of zero-sum trade-offs between different capabilities. These trade-offs seem to be contingent upon various situations

http://mc.manuscriptcentral.com/tprs Email: ijpr@lboro.ac.ukInternational Journal of Production Research

Figure3can also be understood as an ideal "picture" of a single firm as it advances in the development of two capabilities that observe both "cumulative capabilities" and (frontier) "trade-offs", as implied by several authors.


MD Peter C Van Den Akker
email: p.c.van.den.akker@medgen.umcg.nl

Jemima E Mellerio

Anna E Martinez

Lu Liu

Rowdy Meijer

Patricia Jc Dopping-Hepenstal

Anthonie J Van Essen

Hans Scheffer

Robert Mw Hofstra

John A Mcgrath

Patricia J C Dopping-Hepenstal

Robert M W Hofstra

Marcel F Jonkman

The inversa type of recessive dystrophic epidermolysis bullosa is caused by specific arginine and glycine substitutions in type VII collagen

Keywords: recessive dystrophic epidermolysis bullosa, inversa type, genotype-phenotype correlations, type VII collagen, COL7A1

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

INTRODUCTION

The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I; OMIM #226600) is one of the rare variants of dystrophic epidermolysis bullosa. [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF] RDEB-I was first described by Prof. T. Gedde-Dahl Jr in 1971 and is characterized by generalized blistering from birth and during early infancy. [START_REF] Jr | Epidermolysis Bullosa: A clinical, genetic and epidemiological study[END_REF] Later in infancy or childhood, the predilection sites for blistering change and the body flexures, axis and mucous membranes become the predominant sites of blistering. [START_REF] Jr | Epidermolysis Bullosa: A clinical, genetic and epidemiological study[END_REF][START_REF]Epidermolysis Bullosa[END_REF] Neither the cause of the unusual distribution of blistering in RDEB-I, nor the cause of the "transition" from a generalized to inversa distribution is known. The specific age at which this transition in blistering predilection sites takes place is also not known, but it probably varies between patients. As with the main subtypes of RDEB, there is a large clinical variation between inversa patients.

Immunohistochemically, type VII collagen (Col7) is either slightly reduced or normally present in the skin of RDEB-I patients. Anchoring fibrils, which are composed of large numbers of Col7 dimers, can be either reduced or normal in number, and can have a hypoplastic or normal ultrastructural appearance. [START_REF] Bruckner-Tuderman | Type VII collagen is expressed but anchoring fibrils are defective in dystrophic epidermolysis bullosa inversa[END_REF][START_REF] Bruckner-Tuderman | Anchoring fibrils, collagen VII, and neutral metalloproteases in recessive dystrophic epidermolysis bullosa inversa[END_REF][START_REF] Lin | Dystrophic epidermolysis bullosa inversa: report of two cases with further correlation between electron microscopic and immunofluorescence studies[END_REF] Transient retention of Col7 in basal keratinocytes can be a typical immunohistochemical feature in the early stages. [START_REF] Jr | Neonatal retention of type VII collagen, transient bullous dermolysis of the newborn and recessive epidermolysis bullosa dystrophica inversa[END_REF] Similar to the other subtypes of DEB, RDEB-I is caused by mutations in the COL7A1 gene encoding type VII collagen. [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF] However, while genotype-phenotype correlations for the major subtypes of RDEB have been extensively studied in several populations, [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF][START_REF] Hovnanian | Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation[END_REF][START_REF] Gardella | Genotype-phenotype correlation in Italian patients with dystrophic epidermolysis bullosa[END_REF][START_REF] Kern | Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa[END_REF][START_REF] Dang | Review of collagen VII sequence variants found in Australasian patients with dystrophic epidermolysis bullosa reveals nine novel COL7A1 variants[END_REF][START_REF] Varki | Epidermolysis bullosa. II. Type VII collagen mutations and phenotype-genotype correlations in the dystrophic subtypes[END_REF][START_REF] Kern | Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort[END_REF][START_REF] Van Den Akker | Long-term follow-up of patients with recessive dystrophic epidermolysis bullosa in the Netherlands: expansion of the mutation database and unusual phenotype-genotype correlations[END_REF][START_REF] Escámez | The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation[END_REF][START_REF] Jerabkova | Analysis of the COL7A1 gene in Czech patients with dystrophic epidermolysis bullosa reveals novel and recurrent mutations[END_REF] there is little data on genotype-phenotype correlations for RDEB-I. Our study covers the largest RDEB-I cohort reported to date, comprising 20 RDEB-I patients from the Netherlands and the UK. Here we describe this cohort and the genotypes identified in 18 of them, and we discuss the genotype-phenotype correlations identified in these patients and those previously reported in the literature.

PATIENTS AND METHODS

Patients

A diagnosis of DEB was established in a set of 20 patients on the basis of clinical findings, family history, histopathology, immunofluorescence antigen mapping, electron microscopy, and COL7A1 mutation analysis. The diagnosis of RDEB-I was based on the typical clinical findings. [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF][START_REF]Epidermolysis Bullosa[END_REF] All patients were evaluated by the same professionals in the University Medical Center Groningen, the Netherlands (MFJ), Great Ormond Street Hospital for Children or St. Thomas' Hospital, London, UK (JEM, AEM, JAM). Informed consent was given for all skin biopsies and venapunctures, and all studies were approved by the local medical ethical committees (IRB) and conducted in accordance with the guidelines stated in the Declaration of Helsinki.

Immunofluorescence and electron microscopy studies

For immunofluorescence studies (IF), 4 mm punch biopsies of rubbed perilesional skin and/or unaffected skin from non-predilection sites (i.e. the ventral side of the upper arm) were obtained from patients 1-6 and 16 and snap frozen. Four or five µm thick cryostat sections were prepared and the amount of Col7 was determined using monoclonal antibody LH7:2 as previously described. [START_REF] Van Den Akker | Long-term follow-up of patients with recessive dystrophic epidermolysis bullosa in the Netherlands: expansion of the mutation database and unusual phenotype-genotype correlations[END_REF][START_REF] Jonkman | Expression of integrin alpha 6 beta 4 in junctional epidermolysis bullosa[END_REF][START_REF] Groves | A homozygous nonsense mutation within the dystonin gene coding for the coiled-coil domain of the epithelial isoform of BPAG1 underlies a new subtype of autosomal recessive epidermolysis bullosa simplex[END_REF] For electron microscopy, 2 mm punch biopsies were taken from the same locations from the same patients and prepared and analyzed as previously described.

COL7A1 mutation analysis

The COL7A1 gene was fully analyzed for pathological mutations either by complete sequencing or by pre-screening using either Denaturing Gradient Gel Electrophoresis or Conformation-sensitive Capillary Electrophoresis followed by targeted sequencing as described previously. [START_REF] Van Den Akker | Long-term follow-up of patients with recessive dystrophic epidermolysis bullosa in the Netherlands: expansion of the mutation database and unusual phenotype-genotype correlations[END_REF][START_REF] Van Den Akker | Design and validation of a conformation-sensitive capillary electrophoresis system for mutation identification of the COL7A1 gene with automated peak comparison[END_REF] Genomic DNA was extracted from peripheral leukocytes.

RESULTS

Clinical characteristics

In total, we identified 20 patients with RDEB-I, six from the Netherlands (patients 1-6) and 14 from the UK (patients 7-20). Clinical characteristics are listed in supplemental Table S1 and Figure 1. Patients 1 and 2 are sister and brother; patient 14 reported two similarly affected sibs, but they are not known in our clinics; no other patients had any siblings affected with DEB. Eighteen patients (90%) presented with generalized blistering and skin erosions at birth or shortly after and during early infancy; in one the onset of blistering was within the first six months and in another it was unknown. Seven (35%) had congenital localized absence of skin (CLAS), mostly on the lower legs/ankles/feet, which remained fragile during life. With age, blistering on the extremities decreased markedly in most patients and became more restricted to the body trunk and folds. The most frequently affected skin areas were the proximal body flexures, i.e. the axillae and groins, and the body axis, i.e. the back, neck and buttocks symmetrically. None of the patients had involvement of the acra as the dominant feature. In fact, even after trauma most patients only occasionally experienced blistering of hands and feet, and none of them developed 'mitten deformities' as seen in the 'severe generalized' RDEB (RDEB-sev gen) subtype. However, four patients [START_REF]Epidermolysis Bullosa[END_REF][START_REF] Gardella | Genotype-phenotype correlation in Italian patients with dystrophic epidermolysis bullosa[END_REF][START_REF] Van Den Akker | Design and validation of a conformation-sensitive capillary electrophoresis system for mutation identification of the COL7A1 gene with automated peak comparison[END_REF][START_REF] Bruckner-Tuderman | Hereditary skin diseases of anchoring fibrils[END_REF] had more extensive blistering of the acra and developed mild pseudosyndactyly in one of the interdigital web spaces. In contrast, in four patients [START_REF] Jr | Neonatal retention of type VII collagen, transient bullous dermolysis of the newborn and recessive epidermolysis bullosa dystrophica inversa[END_REF][START_REF] Kern | Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort[END_REF][START_REF] Escámez | The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation[END_REF][START_REF] Jerabkova | Analysis of the COL7A1 gene in Czech patients with dystrophic epidermolysis bullosa reveals novel and recurrent mutations[END_REF] skin blistering nearly ceased completely after infancy and they only had mucosal involvement and nail dystrophy.

In 6/20 (30%) patients, lesions of the oral mucosa were already present at birth, whereas in the other patients the oral mucosa became affected from infancy onwards (Table S1). When the RDEB-I phenotype had established, all patients had severe mucosal involvement, mostly affecting the oral cavity, oesophagus and anus. In 6/10 (60%) women the genital skin and mucosa were affected, and in one this severely complicated healing after childbirth. Swallowing difficulty or dysphagia was reported in 17/20 (85%) patients. Twelve patients (60%) had proven oesophageal strictures and 10 (50%) needed repeated oesophageal dilatations; patient 5 received more than 150 dilatations before the age of 30. Ankyloglossia and microstomia due to scarring of the oral mucosa were present in 15 (75%) and 13 (65%) patients, respectively, and was noted as early as 6 years of age (patient 15). Blistering of the external auditory canal was seen in 7/20 (35%) patients, which was complicated by narrowing of the canal in three, and subsequent conductive hearing loss after the age of 30 in two (10%).

None of the patients were growth retarded, had renal failure or developed squamous cell carcinomas. Patients 10, 12, 15, and 20 had anaemic periods and patient 12 required intermittent blood transfusions. Constipation was an almost invariable feature, although only in part due to anal blistering.

Transition age

The age of transition of blistering from extremities to body axis and flexures was reported in 13/20 patients and ranged from one to 18 years. In nine patients, transition was reported between birth and age four years. Patient 6 was the only one in whom the transition age could be objectively determined. He was diagnosed with probable recessive bullous dermolysis of the newborn, since his generalized blisters present at birth disappeared within the first few months, and the immunofluorescence biopsy revealed Col7 retention in basal keratinocytes.

However, towards the end of his first year, blistering activity increased on the trunk, axillae, intraorally and especially in the diaper area. Hence, transition age was before the end of the first year.

Immunofluorescence and electron microscopy findings

Immunofluorescence studies on healthy skin from non-predilection sites revealed normal Col7 in patients 3, 4, 6, 16, and reduced Col7 in patient 1. Retention of Col7 was noted in basal keratinocytes of patient 3 (aged 33) and patient 6, in whose skin both small and large Col7 positive granules were seen at the age of 1 month. Basal keratinocytes of a peri-lesional skin biopsy of patient 2, obtained a few days after birth, also revealed Col7 retention. Perilesional skin from patient 5 revealed reduced Col7. Electron microscopy studies revealed slightly hypoplastic anchoring fibrils in normal (patient 4) or reduced numbers (patients 3, 6, 16) at non-predilection sites. Electron microscopy studies of affected skin revealed a reduced number of abnormal or rudimentary anchoring fibrils in patients 1, 2, 3, 4, 5, and 16. Col7containing vesicles were seen in the rough endoplasmic reticulum in patient 2.

Genotypes and genotype-phenotype correlations

Genotypes were available for 18 of our 20 RDEB-I patients. The genotypes of patients 1-5 have been reported previously. [START_REF] Van Den Akker | Long-term follow-up of patients with recessive dystrophic epidermolysis bullosa in the Netherlands: expansion of the mutation database and unusual phenotype-genotype correlations[END_REF] Table 1 lists the 29 full genotypes associated with RDEB-I identified in this study and the literature. In bold novel COL7A1 mutations.

Six genotypes consist of homozygous or compound heterozygous missense mutations within the triple-helix domain (THD) of Col7. Twenty-three genotypes are composed of THD missense mutations combined with mutations generating a premature truncation codon (PTC)

or an unidentified second mutation (pat 13). Since PTC mutations are generally considered to result in no functional protein, the functional genotype in these patients is determined by the hemizygous missense mutation. [START_REF] Christiano | Premature termination codon mutations in the type VII collagen gene in recessive dystrophic epidermolysis bullosa result in nonsense-mediated mRNA decay and absence of functional protein[END_REF] Hence, the functional genotype in RDEB-I is that of a homozygous, compound heterozygous or hemizygous missense mutation within the THD.

To date, 19 missense mutations have been associated with RDEB-I, all of which are either substitutions of an arginine (ASs, n = 5) or a glycine (GSs, n = 14) (Table 1, Fig. 2).

The 14 GSs are located at 13 different glycine codons. None of the carrier parents were affected, indicating that the mutations are recessive. The question is whether these recessive ASs or GSs are specific for RDEB-I or whether they can also cause other (R)DEB subtypes.

Of the five ASs, the p.Arg2063Gly, p.Arg2622Trp, and p.Arg2628Trp mutations have been exclusively reported in RDEB-I, whereas the p.Arg2008Gly and p.Arg2069Cys mutations were also found in non-inversa RDEB subtypes (Table S2). Nine of the 14 recessive GSs have been exclusively reported in RDEB-I, whereas four GSs were also reported in non-inversa RDEB subtypes and the p.Gly2213Arg mutation was reported as a dominant GS (Table S3).

Supplementary tables S2 and S3, and Fig. 2 show detailed information about the ASs and GSs.

To

DISCUSSION

The cause of the typical 'inverse' distribution of blistering in RDEB-I is not well understood.

We identified 20 Dutch and UK RDEB-I patients and expanded the list of known RDEB-I genotypes from the literature with nine new genotypes, which enabled us to conduct an extensive genotype-phenotype correlation study in RDEB-I. Our results suggest that RDEB-I is caused by a set of specific recessive arginine and glycine substitutions within the THD of Col7.

Clinical characteristics

All 20 patients displayed the typical inversa distribution of skin blistering in the proximal body flexures and the body axis. Mucosal involvement, especially of the oral mucosa, was present in all patients. Nearly all patients started with generalized blistering and a third had congenital localized absence of skin. The age of transition from generalized to inversa distribution took place before the age of 4 years in most patients, but ranged from 1 to 18 years. Marked clinical variation was noted between patients and the clinical severity of RDEB-I seems to cover a spectrum, as seen in other DEB phenotypes. [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF] Four patients had 'mucosal only' RDEB-I in which the mucosal involvement dominated, including severe oesophageal stenosis requiring multiple dilatations, but skin blistering was almost absent. A similar phenotype has been described previously, [START_REF] Kern | Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa[END_REF][START_REF] Zimmer | Esophageal stenosis in childhood: dystrophic epidermolysis bullosa without skin blistering due to collagen VII mutations[END_REF][START_REF] Csikos | High frequency of the 425A-->G splice-site mutation and novel mutations of the COL7A1 gene in central Europe: significance for future mutation detection strategies in dystrophic epidermolysis bullosa[END_REF] but was not considered to be within the RDEB-I spectrum due to absence of typical blistering in the body flexures. We think that patients with 'mucosal only' RDEB form the mildest end of the inversa spectrum (with regard to the skin), because the severe mucosal involvement is not part of the milder forms of RDEB-O. [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF][START_REF] Van Den Akker | Long-term follow-up of patients with recessive dystrophic epidermolysis bullosa in the Netherlands: expansion of the mutation database and unusual phenotype-genotype correlations[END_REF] On the other end of the RDEB-I spectrum, four patients had significant involvement of the acra, leading to mild flexion contractures and limited pseudosyndactyly. This end of the RDEB-I spectrum might be difficult to distinguish from RDEB-O or even RDEB-sev gen. [START_REF] Bruckner-Tuderman | Anchoring fibrils, collagen VII, and neutral metalloproteases in recessive dystrophic epidermolysis bullosa inversa[END_REF][START_REF] Jr | Epidermolysis bullosa: A comprehensive review of classification, management and laboratory studies[END_REF] However, these RDEB-I patients were not growth retarded and none had developed squamous cell carcinoma.

A diagnosis of RDEB-I has important implications for the patient and clinician, especially when skin blistering is relatively mild. Nearly all patients have severe oral and oesophageal blistering resulting in intra-oral scarring, microstomia, ankyloglossia, and oesophageal strictures often requiring repeated dilatations. This can result in decreased food intake, poor dental hygiene and oral infections, which warrant careful follow-up of growth and weight and aggressive dental care. Peri-anal blistering commonly results in constipation that should be asked about carefully and treated if present. In women, genital blistering and scarring can interfere with sexual function, complicate vaginal delivery and recovery afterwards, which may be a reason for elective caesarean section. Moreover, blistering of the external auditory canal may lead to conductive hearing loss, requiring hearing aids after the age of 30. Careful inspection and follow-up of the auditory canals is thus warranted and audiologic evaluation may be indicated at later ages.

Genotype-phenotype correlations

The 29 functional genotypes of our 18 RDEB-I patients and those reported in the literature consist of homozygous, compound heterozygous or hemizygous (i.e. in conjunction with a PTC) recessive ASs or GSs within the THD of Col7. To date, five recessive ASs and 14

recessive GSs have been associated with RDEB-I. The collagenous THD domain is composed of many consecutive Gly-X-Y triplets that form a triple-helix. and carboxyl (COL11-20) regions. The glycine residues are located at the centre of the triplehelix and are essential for stable triple-helix formation, whereas the amino acids at the X and Y positions are exposed on the helix surface and are less essential for triple-helix stability. [START_REF] Persikov | Stability related bias in residues replacing glycines within the collagen triple helix (Gly-Xaa-Yaa) in inherited connective tissue disorders[END_REF][START_REF] Fritsch | Dominant-negative effects of COL7A1 mutations can be rescued by controlled overexpression of normal collagen VII[END_REF] Substitutions of glycine residues therefore have major disruptive effects on triple-helix folding and/or stability, whereas substitutions of X and Y residues not necessarily exert pathogenic effects. Therefore, pathogenicity of the GSs identified in RDEB-I was not surprising. Pathogenicity of the RDEB-I ASs was also likely, as they are located at highly conserved positions within Col7.

Two RDEB-I ASs and five GSs have also been found in patients with phenotypes other than RDEB-I. This observation suggests that some arginine and glycine codons have the potential to result in RDEB-I when mutated, but other as yet unidentified factors determine the resultant clinical phenotype, which can be RDEB-I or sometimes other. The puzzling question remains as to why some specific arginine and glycine codons possess this potential to induce RDEB-I when mutated, since ASs and GSs in general cause phenotypes other than RDEB-I. [START_REF] Dang | Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa[END_REF] We therefore looked more closely into the position of the arginine and glycine codons in the THD and into the nature of the substituting amino acids.

The arginine substitutions

The five arginine codons are located in collagenous subdomains in the carboxyl region of the THD, of which three in the first two subdomains after the hinge region (Fig. 2, Table S2). The position of the arginine within the Gly-X-Y triplet does not differ from arginines involved in other phenotypes: the five RDEB-I arginines reside in the Y position as do 7 of 9 arginines involved in other DEB subtypes.

Our findings suggest that for each of the five arginines there is only a specific substitution that can lead to RDEB-I (Tables S2). The arginine at codon 2063 was found substituted by glycine in RDEB-I, whereas substituted by tryptophan in other RDEB phenotypes; the arginine at codon 2622 was found substituted by tryptophan in RDEB-I, whereas substituted by glutamine in RDEB-HS. The p.Arg2008Gly mutation was found homozygously in RDEB-I, whereas substitution of this arginine by cysteine or histidine was reported in other RDEB subtypes. Of note, the p.Arg2008Gly mutation was also found homozygously in a single RDEB-HS patient, emphasizing that the COL7A1 mutation is not the only factor determining the inversa phenotype. On the other hand, the p.Arg2069Cys mutation was not only found in 12 inversa patients, but also in four with RDEB-O. However, it cannot be excluded that these patients will ultimately develop the RDEB-I phenotype, as it can take years for the inversa phenotype to become evident. Therefore, the p.Arg2069Cys is the most frequently observed RDEB-I mutation and can be considered specific for RDEB-I.

The specific substituting amino acids leading to RDEB-I, however, are different for each arginine codon and from chemically different groups (Table S2). At codons 2008, 2063 and 2069, the large, hydrophilic arginine is substituted by the smaller, nonpolar residues, glycine or cysteine, whereas at codons 2622 and 2628 the arginine is substituted by the large, hydrophobic and aromatic tryptophan. The same substituting amino acids have been found to replace other arginines in the THD in non-inversa phenotypes (Fig 2). This emphasizes that there is not a specific type of amino acid that leads to RDEB-I when replacing an arginine, but it is the combination of a specific substituting amino acid at a specific arginine codon position.

Interestingly, the arginine codons 2622 and 2628 in COL19 are located close to the cysteine residue at position 2634. This is the only cysteine residue in the THD and might be involved in disulfide binding with the non-collagenous domain of a pairing Col7 molecule. [START_REF] Christiano | Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms[END_REF] Substituting either arginine 2622 or 2628 could therefore lead to instable Col7 dimers as a result of local conformational changes.

The glycine substitutions

All 13 glycine codons associated with RDEB-I are located in collagenous subdomains of the THD (Fig. 2, Table S3). Four of 13 (31%) glycine codons are located in subdomain COL19.

This number is surprisingly high, even though COL19 is the largest subdomain, containing almost 16% of Gly-X-Y repeats. These four GSs, as well as p.Gly2775Ser in COL20 are located close to the carboxyl end of the THD. Additionally, the p.Gly1347Trp mutation in the COL2 subdomain is located near the amino end of the THD and glycine codon 1907 (COL10) is located near the hinge region, the end of the amino region of the THD. It can thus be speculated that GSs located at either end of the amino and carboxyl regions of the THD can cause the relatively mild RDEB-I phenotype. This agrees with the finding that GSs near the ends of the THD cause less severe triple-helix destabilization than central GSs and concordantly lead to less severe disease. [START_REF] Zimmer | Esophageal stenosis in childhood: dystrophic epidermolysis bullosa without skin blistering due to collagen VII mutations[END_REF] Interestingly, the amino acids replacing the glycines are either chemically very different and destabilizing, or chemically quite similar and less destabilizing. [START_REF] Persikov | Stability related bias in residues replacing glycines within the collagen triple helix (Gly-Xaa-Yaa) in inherited connective tissue disorders[END_REF] Recently, Chiaverini et al. suggested that GSs located near the borders of collagenous subdomains exert different, i.e. milder, pathogenic effects than GSs located in the centre of collagenous subdomains and consequently result in RDEB-I. [START_REF] Chiaverini | Inversa Dystrophic Epidermolysis Bullosa Is Caused by Missense Mutations at Specific Positions of the Collagenic Domain of Collagen Type VII[END_REF] However, the combined genotypic data of our RDEB-I patients and those reported in the literature show that of the 13 glycine codons involved in RDEB-I only 6/13 (46%) are located in the first three or last three

Gly-X-Y triplets of their respective collagenous subdomain. The other 7/13 (54%) are located in the central portion of the subdomain (Table S3). Hence, a GSs location near the border of a collagenous subdomain might predispose to RDEB-I, but it is certainly not the only factor, since specific central GSs also have the potential to induce RDEB-I.

There were no differences in the location of the glycine codons or the nature of the substituting amino acids between GSs causing the 'mucosal only' phenotype and the phenotype with limited acral involvement (Tables 1 andS3). However, the four glycine codons involved in RDEB-I with limited acral involvement have been reported in the RDEBsev gen or DDEB phenotypes. In contrast, of the glycine codons involved in the RDEB-I 'mucosal only' phenotype only glycine codon 1347 was reported in other, milder RDEB phenotypes. [START_REF] Kern | Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa[END_REF][START_REF] Terracina | Compound heterozygosity for a recessive glycine substitution and a splice site mutation in the COL7A1 gene causes an unusually mild form of localized recessive dystrophic epidermolysis bullosa[END_REF][START_REF] Schumann | Dystrophic epidermolysis bullosa pruriginosa is not associated with frequent FLG gene mutations[END_REF] Putative temperature related pathophysiology in RDEB-I

Because the lesions in RDEB-I are mainly restricted to body locations where skin temperatures are the highest (Fig. 3), [START_REF] Herry | Quantitative assessment of pain-related thermal dysfunction through clinical digital infrared thermal imaging[END_REF][START_REF] Oji | Bathing suit ichthyosis is caused by transglutaminase-1 deficiency: evidence for a temperature-sensitive phenotype[END_REF] we hypothesize that the pathophysiology in RDEB-I is temperature-dependent. This leads to increased Col7 destabilization with subsequent blistering only in these body regions and not on the more distal body parts where skin temperatures are generally lower.

The invariable involvement of the oral mucosa and oesophageal involvement in most patients supports this hypothesis. Temperature-dependent pathophysiology has been shown for bathing suit ichthyosis (BSI), in which skin distribution is quite similar to RDEB-I. [START_REF] Oji | Bathing suit ichthyosis is caused by transglutaminase-1 deficiency: evidence for a temperature-sensitive phenotype[END_REF] Moreover, also in BSI the skin features are more severe in infancy than later on. In BSI, specific mutations in the transglutaminase-1 gene, TGM1, lead to decreased enzyme activity at temperatures above 33°C, [START_REF] Aufenvenne | Transglutaminase-1 and bathing suit ichthyosis: molecular analysis of gene/environment interactions[END_REF] explaining the 'inversa-like' skin distribution. Interestingly, most BSI causing TGM1 mutations found to date are arginine substitutions. [START_REF] Hackett | Genotype-phenotype correlations with TGM1: clustering of mutations in the bathing suit ichthyosis and selfhealing collodion baby variants of lamellar ichthyosis[END_REF] The ASs found in RDEB-I could thus exert the same effects as the GSs. A temperature-dependent effect in skin disorders has also been noted for mutations in the TYR and CDKN2A genes. [START_REF] Berson | A common temperaturesensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature[END_REF][START_REF] Florell | Longitudinal assessment of the nevus phenotype in a melanoma kindred[END_REF] Temperature-dependent pathophysiology could also explain the transition from generalized to inversa distribution, as one can assume that skin temperature in utero will be higher than postnatally in all body regions. An obvious question is whether patients have more severe blistering during warmer circumstances, but this was denied by all patients.

Hence, the temperature hypothesis is an interesting topic for future studies.

Conclusion and clinical implications

Our results suggest that RDEB-I is caused by specific recessive arginine and glycine substitutions in the THD of Col7. The p.Arg2069Cys mutation is the most frequently observed RDEB-I mutation. This study provides detailed insights into genotype-phenotype correlations in RDEB-I, which is a first step towards understanding the pathophysiology of the typical inverse distribution in this rare RDEB subtype. The exact mechanisms by which these ASs and GSs cause RDEB-I are not yet understood, but we hypothesize that the pathophysiology of the inversa distribution is temperature-dependent.

These findings have important implications for the diagnosis and prognosis in DEB,

since the identification of one of the specific ASs or GSs as the only functional allele in a newborn with DEB is predictive of the future development of RDEB-I. However, a phenotype with more severe skin involvement may occasionally develop due to currently unknown genetic, epigenetic or non-genetic modifiers.

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  check whether ASs in general might cause the RDEB-I phenotype, we studied the phenotypes of patients carrying other ASs. The THD of Col7 contains a total of 94 arginines in Gly-X-Y repeats,[35] of which 12 have been found substituted in DEB (HGMD professional database, www.hgmd.org, and unpublished data (HS), 26 August 2010). Substitutions of 5/12 arginines (42%) are involved in RDEB-I, whereas substitutions of 9/12 arginines (75%) are involved in other DEB phenotypes. Hence, it seems unlikely that ASs are in general the cause of RDEB-I.

[ 35 ]

 35 These Gly-X-Y repeat sequences are separated into 20 collagenous subdomains (COL1-20) by 19 interruptions or imperfections (IM1-19) that provide flexibility to the protein. The largest imperfection, the hinge (IM10), is composed of 39 amino acids and divides the THD into amino (COL1-10)

Figure 1 .

 1 Figure 1. Clinical features of patients with RDEB-inversa.

Figure 2 .

 2 Figure 2. Arginine and glycine substitutions associated with RDEB-I.

Figure 3 .

 3 Figure 3. Digital thermographs of a healthy human body.

Table 1 . Genotypes of RDEB-inversa patients reported in this study and in the literature. 1 No. Mutation 1 1 Mutation 2 1 Patients Reference Nucleotide Amino acid Exon Consequence 2 Domain 3 Nucleotide 4 Amino acid Exon 5 Consequence 2 Domain 3

 1

	c.4039G>T	p.Gly1347Trp 34	Missense	THD	c.8540insC	p.Glu2848X	116	PTC	NC2	Pat 16	This study
	c.4945C>G	p.Gly1649Arg 53	Missense	THD	NF 6					Pat 13	This study
	c.5282G>C	p.Gly1761Ala 60	Missense	THD	c.5282G>C	p.Gly1761Ala	60	Missense	THD	Pat 6	This study
	c.5720G>A	p.Gly1907Glu 68	Missense	THD	c.7805G>A	p.Gly2602Glu	105	Missense	THD	Pat 3	[14]
	c.5720_5721	p.Gly1907Asp 68	Missense	THD	c.4018C>T	p.Arg1340X	34	PTC	THD	2x	[26]
	GA>AT
	c.5720_5721	p.Gly1907Asp 68	Missense	THD	c.5047C>T	p.Arg1687X	54	PTC	THD	Pat 12,	This study
	GA>AT										17
	c.6022C>G	p.Arg2008Gly 73	Missense	THD	c.6022C>G	p.Arg2008Gly	73	Missense	THD	2x	[20, 21]
	c.6187C>G	p.Arg2063Gly 74	Missense	THD	c.706C>T	p.Arg236X	6	PTC	NC1	1x	[8]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.425A>G	p.Lys142Arg	3	Splice PTC NC1	3x	[20, 24]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.3551-3T>G		IVS26 Splice PTC NC1	Pat 5	[14]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.3894+1G>A		IVS30 Splice PTC THD	1x	[16]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.3895-1G>A		IVS30 Splice PTC THD	1x	[25]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.4018C>T	p.Arg1340X	34	PTC	THD	1x	[16]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.4027C>T	p.Arg1343X	34	PTC	THD	1x	[16]
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.5662insG	p.Pro1888AlafsX27 67	PTC	THD	Pat 11	This Study
	c.6205C>T	p.Arg2069Cys 74	Missense	THD	c.6205C>T	p.Arg2069Cys	74	Missense	THD	3x (pat	This study, [14]
											4, 8, 18)
	c.6262G>A	p.Gly2088Arg 75	Missense	THD	c.676C>T	p.Arg226X	5	PTC	NC1	1x	[26]
	c.6637G>A	p.Gly2213Arg 83	Missense	THD	c.4918delG	p.Gly1640ValfsX70 52	PTC	THD	Pat 9	This study
	c.6815G>C	p.Gly2272Ala 86	Missense	THD	c.1363G>T	p.Gln455X	11	PTC	NC1	Pat 15	This study
	c.7415G>A	p.Gly2472Asp 97	Missense	THD	c.1874delGT	p.Ser625ArgfsX5	14	PTC	NC1	1x	[26]
	c.7864C>T	p.Arg2622Trp 105	Missense	THD	c.553C>T	p.Arg185X	5	PTC	NC1	1x	[15]
	c.7864C>T	p.Arg2622Trp 105	Missense	THD	c.2482delCT	p.Ser828CysfsX37	19	PTC	NC1	1x	[9]
	c.7882C>T	p.Arg2628Trp 106	Missense	THD	c.1332C>A	p.Tyr444X	10	PTC	NC1	1x	[26]
	c.8065G>A	p.Gly2689Arg 109	Missense	THD	c.1732C>T	p.Arg578X	13	PTC	NC1	Pat 7	This study
	c.8065G>C	p.Gly2689Arg 109	Missense	THD	c.6527insC	p.Gly2177TrpfsX113 80	PTC	THD	1x	[10]
	c.8083G>A	p.Gly2695Ser	109	Missense	THD	c.8083G>A	p.Gly2695Ser	109	Missense	THD	Pat 1, 2	[12, 14, 22]
	c.8156G>C	p.Gly2719Ala 110	Missense	THD	c.2051-1G>C		IVS15 Splice PTC NC1	Pat 19	This study
	c.8323G>A	p.Gly2775Ser	112	Missense	THD	c.425A>G	p.Lys142Arg	3	Splice PTC NC1	2x (pat	This study, [23]
											14)
	c.8323G>A	p.Gly2775Ser	112	Missense	THD	c.8323G>A	p.Gly2775Ser	112	Missense	THD	1x	[26]

  Kern et al. showed by in vitro experiments that Col7 the recessive GSs found in RDEB-I only slightly lower the temperature dissociation thresholds for Col7 homotrimers. The only body regions where skin temperatures are continuously high enough to cross these temperature thresholds are the inversa regions.

	homotrimers from a 'mucosal only' patient (genotype 18, Table 1) dissociated at lower
	temperatures (Tm = 33-35°C) compared to normal Col7 (Tm = 39-41°C).[10] Moreover,
	Fritsch et al. showed that Col7 trimers harbouring three different dominant GSs in the THD
	had even more reduced thermostability (Tm 20-33°C, mono-expression).[39] Furthermore,
	four GSs in the THD of type XVII collagen also reduced the thermostability of recombinant
	proteins.[42] This reduced thermostability reflects folding defects of procollagen molecules
	into Col7 trimers and/or packing defects of Col7 trimers into anchoring fibrils. The lower
	dissociation temperatures of Col7 homotrimers harbouring the dominant GSs suggest that
	dominant GSs have more dramatic destabilizing effects on Col7 than recessive GSs. Hence,
	we speculate that

PTC, premature termination codon; splice PTC, splice site affecting mutation resulting in PTC.

THD, triple-helix domain; NC1, non-collagenous 1 domain; NC2, non-collagenous 2 domain.

NF, not found; NA, not available.

IVS, intervening sequences (i.e. introns)

Only a single mutation could be identified despite complete sequencing of the other allele.

ACKNOWLEDGEMENTS

We would like to thank the patients and their parents for their cooperation and Jackie Senior for editing the manuscript. This study was supported by the Netherlands Organization for Health Research and Development (ZonMw) grant 92003541, the Prof. J.P. Nater Foundation and the Vlinderkind (Dutch Butterfly) Foundation. JEM and JAM acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust.

COMPETING INTERESTS

None.