Functional modularity and mechanical stress shape plastic responses during fish development.

The adaptive potential of plastic phenotypes relies on combined developmental responses. We investigated how manipulation of developmental conditions related to foraging mode in the fish Megaleporinus macrocephalus induces plastic responses at different levels: 1) functional modularity of skull bones, 2) biomechanical properties of the chondrocranium using Finite Element Models, 3) bmp4 expression levels, used as a proxy for molecular pathways involved in bone responses to mechanical load. We identified new modules in experimental groups, suggesting increased integration in specific head bone elements associated with the development of subterminal and upturned mouths, which are major features of Megaleporinus plastic morphotypes released in the lab. Plastic responses in head shape involved differences in the magnitude of mechanical stress, which seem restricted to certain chondrocranium regions. Three bones represent a 'mechanical unit' related to changes in mouth position induced by foraging mode, suggesting that functional modularity might be enhanced by the way specific regions respond to mechanical load. Differences in bmp4 expression levels between plastic morphotypes indicate associations between molecular signaling pathways and biomechanical responses to load. Our results offer a multilevel perspective of epigenetic factors involved in plastic responses, expanding our knowledge about mechanisms of developmental plasticity that originate novel complex phenotypes.

Bridging Performance and Adaptive Landscapes to Understand Long-Term Functional Evolution.

AbstractUnderstanding functional adaptation demands an integrative framework that captures the complex interactions between form, function, ecology, and evolutionary processes. In this review, we discuss how to integrate the following two distinct approaches to better understand functional evolution: (1) the adaptive landscape approach (ALA), aimed at finding adaptive peaks for different ecologies, and (2) the performance landscape approach (PLA), aimed at finding performance peaks for different ecologies. We focus on the Ornstein-Uhlenbeck process as the evolutionary model for the ALA and on biomechanical modeling to estimate performance for the PLA. Whereas both the ALA and the PLA have each given insight into functional adaptation, separately they cannot address how much performance contributes to fitness or whether evolutionary constraints have played a role in form-function evolution. We show that merging these approaches leads to a deeper understanding of these issues. By comparing the locations of performance and adaptive peaks, we can infer how much performance contributes to fitness in species' current environments. By testing for the relevance of history on phenotypic variation, we can infer the influence of past selection and constraints on functional adaptation. We apply this merged framework in a case study of turtle shell evolution and explain how to interpret different possible outcomes. Even though such outcomes can be quite complex, they represent the multifaceted relations among function, fitness, and constraints.

Can extreme climatic events induce shifts in adaptive potential? A conceptual framework and empirical test with Anolis lizards.

Multivariate adaptation to climatic shifts may be limited by trait integration that causes genetic variation to be low in the direction of selection. However, strong episodes of selection induced by extreme climatic pressures may facilitate future population-wide responses if selection reduces trait integration and increases adaptive potential (i.e., evolvability). We explain this counter-intuitive framework for extreme climatic events in which directional selection leads to increased evolvability and exemplify its use in a case study. We tested this hypothesis in two populations of the lizard Anolis scriptus that experienced hurricane-induced selection on limb traits. We surveyed populations immediately before and after the hurricane as well as the offspring of post-hurricane survivors, allowing us to estimate both selection and response to selection on key functional traits: forelimb length, hindlimb length, and toepad area. The direct selection was parallel in both islands and strong in several limb traits. Even though overall limb integration did not change after the hurricane, both populations showed a non-significant tendency toward increased evolvability after the hurricane despite the direction of selection not being aligned with the axis of most variance (i.e., body size). The population with comparably lower between-limb integration showed a less constrained response to selection. Hurricane-induced selection, not aligned with the pattern of high trait correlations, likely conflicts with selection occurring during normal ecological conditions that favours functional coordination between limb traits, and would likely need to be very strong and more persistent to elicit a greater change in trait integration and evolvability. Future tests of this hypothesis should use G-matrices in a variety of wild organisms experiencing selection due to extreme climatic events.

Sex-specific multivariate morphology/performance relationships in Anolis carolinensis.

Animals rely on their ability to perform certain tasks sufficiently well to survive, secure mates and reproduce. Performance traits depend on morphology, and so morphological traits should predict performance, yet this relationship is often confounded by multiple competing performance demands. Males and females experience different selection pressures on performance, and the consequent sexual conflict over performance expression can either constrain performance evolution or drive sexual dimorphism in both size and shape. Furthermore, change in a single morphological trait may benefit some performance traits at the expense of others, resulting in functional trade-offs. Identifying general or sex-specific relationships between morphology and performance at the organismal level thus requires a multivariate approach, as individuals are products of both an integrated phenotype and the ecological environment in which they have developed and evolved. We estimated the multivariate morphology→performance gradient in wild-caught, green anoles (Anolis carolinensis) by measuring external morphology and forelimb and hindlimb musculature, and mapping these morphological traits to seven measured performance traits that cover the broad range of ecological challenges faced by these animals (sprint speed, endurance, exertion distance, climbing power, jump power, cling force and bite force). We demonstrate that males and females differ in their multivariate mapping of traits on performance, indicating that sex-specific ecological demands likely shape these relationships, but do not differ in performance integration.

Development and function explain the modular evolution of phalanges in gecko lizards.

Selective regimes favouring the evolution of functional specialization probably affect covariation among phenotypic traits. Phalanges of most tetrapods develop from a conserved module that constrains their relative proportions. In geckos, however, biomechanical specializations associated with adhesive toepads involve morphological variation in the autopodium and might reorganize such modular structures. We tested two hypotheses to explain the modular architecture of hand bones in geckos, one based on developmental interactions and another incorporating functional associations related to locomotion, and compared the empirical support for each hypothetical module between padded and padless lineages. We found strong evidence for developmental modules in most species, which probably reflects embryological constraints during phalangeal formation. Although padded geckos exhibit a functional specialization involving the hyperextension of the distal phalanges that is absent in padless species, the padless species are the ones that show a distal functional module with high integration. Some ancestrally padless geckos apparently deviate from developmental predictions and present a relatively weak developmental module of phalanges and a strongly integrated distal module, which may reflect selective regimes involving incipient frictional adhesion in digit morphology. Modularity of digit elements seems dynamic along the evolutionary history of geckos, being associated with the presence/absence of adhesive toepads.

Detecting patterns of correlational selection with sampling error: A simulation study.

The adoption of a multivariate perspective of selection implies the existence of multivariate adaptive peaks and pervasive correlational selection that promotes co-adaptation between traits. However, to test for the ubiquity of correlational selection in nature, we must first have a sense of how well can we estimate multivariate nonlinear selection (i.e., the γ-matrix) in the face of sampling error. To explore the sampling properties of estimated γ-matrices, we simulated inidividual traits and fitness under a wide range of sample sizes, using different strengths of correlational selection and of stabilizing selection, combined with different number of traits under selection, different amounts of residual variance in fitness, and distinct patterns of selection. We then ran nonlinear regressions with these simulated datasets to simulate γ-matrices after adding random error to individual fitness. To test how well could we detect the imposed pattern of correlational selection at different sample sizes, we measured the similarity between simulated and imposed γ-matrices. We show that detection of the pattern of correlational selection is highly dependent on the total strength of selection on traits and on the amount of residual variance in fitness. Minimum sample size needs to be at least 500 to precisely estimate the pattern of correlational selection. Furthermore, a pattern of selection in which different sets of traits contribute to different functions is the easiest to diagnose, even when using a large number of traits (10 traits), but with sample sizes in the order of 1000 individuals. Consequently, we recommend working with sets of traits from distinct functional complexes and fitness proxies less prone to effects of environmental and demographic stochasticity to test for correlational selection with lower sample sizes.

Correlational selection in the age of genomics.

Ecologists and evolutionary biologists are well aware that natural and sexual selection do not operate on traits in isolation, but instead act on combinations of traits. This long-recognized and pervasive phenomenon is known as multivariate selection, or-in the particular case where it favours correlations between interacting traits-correlational selection. Despite broad acknowledgement of correlational selection, the relevant theory has often been overlooked in genomic research. Here, we discuss theory and empirical findings from ecological, quantitative genetic and genomic research, linking key insights from different fields. Correlational selection can operate on both discrete trait combinations and quantitative characters, with profound implications for genomic architecture, linkage, pleiotropy, evolvability, modularity, phenotypic integration and phenotypic plasticity. We synthesize current knowledge and discuss promising research approaches that will enable us to understand how correlational selection shapes genomic architecture, thereby linking quantitative genetic approaches with emerging genomic methods. We suggest that research on correlational selection has great potential to integrate multiple fields in evolutionary biology, including developmental and functional biology, ecology, quantitative genetics, phenotypic polymorphisms, hybrid zones and speciation processes.

Copulatory function and development shape modular architecture of genitalia differently in males and females.

Genitalia are multitasking structures whose development is mediated by numerous regulatory pathways. This multifactorial nature provides an avenue for multiple sources of selection. As a result, genitalia tend to evolve as modular systems comprising semi-independent subsets of structures, yet the processes that give rise to those patterns are still poorly understood. Here, we ask what are the relative roles of development and function in shaping modular patterns of genitalia within populations and across species of stink-bugs. We found that male genitalia are less integrated, more modular, and primarily shaped by functional demands. In contrast, females show higher integration, lower modularity, and a predominant role of developmental processes. Further, interactions among parts of each sex are more determinant to modularity than those between the sexes, and patterns of modularity are equivalent between and within species. Our results strongly indicate that genitalia have been subjected to sex-specific selection, although male and female genitalia are homologous and functionally associated. Moreover, modular patterns are seemingly constant in the evolutionary history of stink-bugs, suggesting a scenario of multivariate stabilizing selection within each sex. Our study demonstrates that interactions among genital parts of the same sex may be more fundamental to genital evolution than previously thought.

Evolution of a complex phenotype with biphasic ontogeny: Contribution of development versus function and climatic variation to skull modularity in toads.

The theory of morphological integration and modularity predicts that if functional correlations among traits are relevant to mean population fitness, the genetic basis of development will be molded by stabilizing selection to match functional patterns. Yet, how much functional interactions actually shape the fitness landscape is still an open question. We used the anuran skull as a model of a complex phenotype for which we can separate developmental and functional modularity. We hypothesized that functional modularity associated to functional demands of the adult skull would overcome developmental modularity associated to bone origin at the larval phase because metamorphosis would erase the developmental signal. We tested this hypothesis in toad species of the Rhinella granulosa complex using species phenotypic correlation pattern (P-matrices). Given that the toad species are distributed in very distinct habitats and the skull has important functions related to climatic conditions, we also hypothesized that differences in skull trait covariance pattern are associated to differences in climatic variables among species. Functional and hormonal-regulated modules are more conspicuous than developmental modules only when size variation is retained on species P-matrices. Without size variation, there is a clear modularity signal of developmental units, but most species have the functional model as the best supported by empirical data without allometric size variation. Closely related toad species have more similar climatic niches and P-matrices than distantly related species, suggesting phylogenetic niche conservatism. We infer that the modularity signal due to embryonic origin of bones, which happens early in ontogeny, is blurred by the process of growth that occurs later in ontogeny. We suggest that the species differing in the preferred modularity model have different demands on the orbital functional unit and that species contrasting in climate are subjected to divergent patterns of natural selection associated to neurocranial allometry and T3 hormone regulation.

PRONTOX - proton therapy to reduce acute normal tissue toxicity in locally advanced non-small-cell lung carcinomas (NSCLC): study protocol for a randomised controlled trial.

BACKGROUND: Primary radiochemotherapy with photons is the standard treatment for locally advanced-stage non-small cell lung cancer (NSCLC) patients. Acute radiation-induced side effects such as oesophagitis and radiation pneumonitis limit patients' quality of life, and the latter can be potentially life-threatening. Due to its distinct physical characteristics, proton therapy enables better sparing of normal tissues, which is supposed to translate into a reduction of radiation-induced side effects. METHODS/DESIGN: This is a single-centre, prospective, randomised controlled, phase II clinical trial to compare photon to proton radiotherapy up to 66 Gy (RBE) with concomitant standard chemotherapy in patients with locally advanced-stage NSCLC. Patients will be allocated in a 1:1 ratio to photon or proton therapy, and treatment will be delivered slightly accelerated with six fractions of 2 Gy (RBE) per week. DISCUSSION: The overall aim of the study is to show a decrease of early and intermediate radiation-induced toxicity using proton therapy. For the primary endpoint of the study we postulate a decrease of radiation-induced side effects (oesophagitis and pneumonitis grade II or higher) from 39 to 12%. Secondary endpoints are locoregional and distant failure, overall survival and late side effects. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with Identifier NCT02731001 on 1 April 2016.

High evolutionary constraints limited adaptive responses to past climate changes in toad skulls.

Interactions among traits that build a complex structure may be represented as genetic covariation and correlation. Genetic correlations may act as constraints, deflecting the evolutionary response from the direction of natural selection. We investigated the relative importance of drift, selection, and constraints in driving skull divergence in a group of related toad species. The distributional range of these species encompasses very distinct habitats with important climatic differences and the species are primarily distinguished by differences in their skulls. Some parts of the toad skull, such as the snout, may have functional relevance in reproductive ecology, detecting water cues. Thus, we hypothesized that the species skull divergence was driven by natural selection associated with climatic variation. However, given that all species present high correlations among skull traits, our second prediction was of high constraints deflecting the response to selection. We first extracted the main morphological direction that is expected to be subjected to selection by using within- and between-species covariance matrices. We then used evolutionary regressions to investigate whether divergence along this direction is explained by climatic variation between species. We also used quantitative genetics models to test for a role of random drift versus natural selection in skull divergence and to reconstruct selection gradients along species phylogeny. Climatic variables explained high proportions of between-species variation in the most selected axis. However, most evolutionary responses were not in the direction of selection, but aligned with the direction of allometric size, the dimension of highest phenotypic variance in the ancestral population. We conclude that toad species have responded to selection related to climate in their skulls, yet high evolutionary constraints dominated species divergence and may limit species responses to future climate change.

[Approval procedures for clinical trials in the field of radiation oncology]. / Genehmigungsverfahren klinischer Studien im Bereich der Radioonkologie.

BACKGROUND AND PURPOSE: Application of ionizing radiation for the purpose of medical research in Germany needs to be approved by the national authority for radiation protection (Bundesamt für Strahlenschutz, BfS). For studies in the field of radiation oncology, differentiation between use of radiation for "medical care (Heilkunde)" versus "medical research" frequently leads to contradictions. The aim of this article is to provide principle investigators, individuals, and institutions involved in the process, as well as institutional review or ethics committees, with the necessary information for this assessment. Information on the legal frame and the approval procedures are also provided. METHODS: A workshop was co-organized by the German Society for Radiation Oncology (DEGRO), the Working Party for Radiation Oncology (ARO) of the German Cancer Society (DKG), the German Society for Medical Physics (DGMP), and the German Cancer Consortium (DKTK) in October 2013. This paper summarizes the results of the workshop and the follow-up discussions between the organizers and the BfS. RESULTS: Differentiating between "Heilkunde" which does not need to be approved by the BfS and "medical research" is whether the specific application of radiation (beam quality, dose, schedule, target volume, etc.) is a clinically established and recognized procedure. This must be answered by the qualified physician(s) ("fachkundiger Arzt" according to German radiation protection law) in charge of the study and the treatments of the patients within the study, taking into consideration of the best available evidence from clinical studies, guidelines and consensus papers. Among the important parameters for assessment are indication, total dose, and fractionation. Radiation treatments applied outside clinical trials do not require approval by the BfS, even if they are applied within a randomized or nonrandomized clinical trial. The decision-making by the "fachkundigem Arzt" may be supported on request by an opinion given by the DEGRO Expert Committee for clinical trials. CONCLUSION: An important aim for promoting clinical research and patient care in radiation oncology is to further professionalize planning and implementation of clinical trials in this field. Correct assessment, at an early stage, whether a trial needs to be approved by the BfS may reduce unnecessary costs and reduce the time needed for the approval procedure for those trials which need to be assessed by the BfS.

Landmark precision and reliability and accuracy of linear distances estimated by using 3D computed micro-tomography and the open-source TINA Manual Landmarking Tool software.

INTRODUCTION: The wider availability of non-destructive and high-resolution methods, such as micro-computed tomography (micro-CT), has prompted its use in anatomical and morphometric studies. Yet, because of the actual scanning procedure and the processing of CT data by software that renders 3D surfaces or volumes, systematic errors might be introduced in placing landmarks as well as in estimating linear distances. Here we assess landmark precision and measurement reliability and accuracy of using micro-CT images of toad skulls and the TINA Manual Landmarking Tool software to place 20 landmarks and extract 24 linear distances. Landmark precision and linear distances calculated from 3D images were compared to the same landmarks and distances obtained with a 3D digitizer in the same skulls. We also compared landmarks and linear distances in 3D images of the same individuals scanned with distinct filters, since we detected variation in bone thickness or density among the individuals used. RESULTS: We show that landmark precision is higher for micro-CT than for the 3D digitizer. Distance reliability was very high within-methods, but decreased in 20 % when 3D digitizer and micro-CT data were joined together. Still, we did not find any systematic bias in estimating linear distances with the micro-CT data and the between-methods errors were similar for all distances (around 0.25 mm). Absolute errors correspond to about 6.5 % of the distance's means for micro-CT resolutions and 3D digitizer comparisons, and to 3 % for the filter type analysis. CONCLUSIONS: We conclude that using micro-CT data for morphometric analysis results in acceptable landmark precision and similar estimates of most linear distances compared to 3D digitizer, although some distances are more prone to discrepancies between-methods. Yet, caution in relation to the scale of the measurements needs to be taken, since the proportional between-method error is higher for smaller distances. Scanning with distinct filters does not introduce a high level of error and is recommended when individuals differ in bone density.

Upper thermal tolerance plasticity in tropical amphibian species from contrasting habitats: implications for warming impact prediction.

Tropical ectothermic species are currently depicted as more vulnerable to increasing temperatures because of the proximity between their upper thermal limits and environmental temperatures. Yet, the acclimatory capacity of thermal limits has rarely been measured in tropical species, even though they are generally predicted to be smaller than in temperate species. We compared critical thermal maximum (CTmax) and warming tolerance (WT: the difference between CTmax and maximum temperature, Tmax), as well as CTmax acclimatory capacity of toad species from the Atlantic forest (AF) and the Brazilian Caatinga (CAA), a semi-arid habitat with high temperatures. Acclimation temperatures represented the mean temperatures of AF and CAA habitats, making estimates of CTmax and WT more ecologically realistic. CAA species mean CTmax was higher compared to AF species in both acclimation treatments. Clutches within species, as well as between AF and CAA species, differed in CTmax plasticity and we discuss the potential biological meaning of these findings. We did not find a trade-off between absolute CTmax and CTmax plasticity, indicating that species can have both high CTmax and high CTmax plasticity. Although CTmax was highly correlated to Tmax, CTmax plasticity was not related to Tmax or Tmax coefficients of variation. CAA species mean WT was lower than for AF species, but still very high for all species, diverging from other studies with tropical species. This might be partially related to over-estimation of vulnerability due to under-appreciation of realistic acclimation treatments in CTmax estimation. Thus, some tropical species might not be as vulnerable to warming as previously predicted if CTmax is considered as a shifting population parameter.

Creating a data exchange strategy for radiotherapy research: towards federated databases and anonymised public datasets.

Disconnected cancer research data management and lack of information exchange about planned and ongoing research are complicating the utilisation of internationally collected medical information for improving cancer patient care. Rapidly collecting/pooling data can accelerate translational research in radiation therapy and oncology. The exchange of study data is one of the fundamental principles behind data aggregation and data mining. The possibilities of reproducing the original study results, performing further analyses on existing research data to generate new hypotheses or developing computational models to support medical decisions (e.g. risk/benefit analysis of treatment options) represent just a fraction of the potential benefits of medical data-pooling. Distributed machine learning and knowledge exchange from federated databases can be considered as one beyond other attractive approaches for knowledge generation within "Big Data". Data interoperability between research institutions should be the major concern behind a wider collaboration. Information captured in electronic patient records (EPRs) and study case report forms (eCRFs), linked together with medical imaging and treatment planning data, are deemed to be fundamental elements for large multi-centre studies in the field of radiation therapy and oncology. To fully utilise the captured medical information, the study data have to be more than just an electronic version of a traditional (un-modifiable) paper CRF. Challenges that have to be addressed are data interoperability, utilisation of standards, data quality and privacy concerns, data ownership, rights to publish, data pooling architecture and storage. This paper discusses a framework for conceptual packages of ideas focused on a strategic development for international research data exchange in the field of radiation therapy and oncology.

Persistent activation of Akt or ERK prevents the toxicity induced by saturated and polyunsaturated fatty acids in RINm5F ß-cells.

The aim of this study was to investigate whether the toxicity of saturated and polyunsaturated fatty acids (PUFA) on RINm5F cells is related to the phosphorylation state of Akt, ERK and PKC delta. The regulation of these kinases was compared in three experimental designs: (a) 4h-exposure, (b) 4h-exposure and a subsequent withdrawn of the FA for a 20 h period and (c) 24h-exposure. Saturated and PUFA were toxic to RINm5F cells even at low concentrations. Also, evidence is provided for a late (i.e. the effect only appeared hours after the treatment) and a persistent regulation (i.e. maintenance of the effect for several hours) of Akt, ERK and PKC delta phosphorylation by the FA. Late activation of PKC delta seems important for palmitate cytotoxicity. Persistent activation of the survival proteins Akt and ERK by stearate, oleate and arachidonate might play an important role to prevent the toxic effect of posterior PKC delta activation. The results shown may explain why a short-period exposure to FA is not enough to induce cytotoxicity in pancreatic beta-cells, since survival pathways are activated. Besides, when this activation is persistent, it may overcome a posterior induction of death pathways.

[Setting up an interdisciplinary cancer center between the poles of internal and external interests]. / Etablierung eines interdisziplinären Krebszentrums im Spannungsfeld interner und externer Interessen.

Excellence in oncology requires specialized centers covering a broad spectrum of oncological competence and technology. Such Comprehensive Cancer Centers, which in most cases are affiliated with a university, are well established in many countries, particularly North America. But despite their advantages, only few of these interdisciplinary cancer centers have so far been set up in Germany. The establishment of a Comprehensive Cancer Center covering patient care, cancer research as well as education and training in Germany will be discussed using the example of the Dresden University Cancer Center. Consideration will be given to the interests of the different groups involved and to critical success factors such as its mission, interdisciplinary leadership structures, interfaces, responsibilities and quality management.

Conspicuous involvement of desmin tail mutations in diverse cardiac and skeletal myopathies.

Myofibrillar myopathy (MFM) encompasses a genetically heterogeneous group of human diseases caused by mutations in genes coding for structural proteins of muscle. Mutations in the intermediate filament (IF) protein desmin (DES), a major cytoskeletal component of myocytes, lead to severe forms of "desminopathy," which affects cardiac, skeletal, and smooth muscle. Most mutations described reside in the central alpha-helical rod domain of desmin. Here we report three novel mutations--c.1325C>T (p.T442I), c.1360C>T (p.R454W), and c.1379G>T (p.S460I)--located in desmin's non-alpha-helical carboxy-terminal "tail" domain. We have investigated the impact of these and four--c.1237G>A (p.E413K), c.1346A>C (p.K449T), c.1353C>G (p.I451M), and c.1405G>A (p.V469M)--previously described "tail" mutations on in vitro filament formation and on the generation of ordered cytoskeletal arrays in transfected myoblasts. Although all but two mutants (p.E413K, p.R454W) assembled into IFs in vitro and all except p.E413K were incorporated into IF arrays in transfected C2C12 cells, filament properties differed significantly from wild-type desmin as revealed by viscometric assembly assays. Most notably, when coassembled with wild-type desmin, these mutants revealed a severe disturbance of filament-formation competence and filament-filament interactions, indicating an inherent incompatibility of mutant and wild-type protein to form mixed filaments. The various clinical phenotypes observed may reflect altered interactions of desmin's tail domain with different components of the myoblast cytoskeleton leading to diminished biomechanical properties and/or altered metabolism of the individual myocyte. Our in vitro assembly regimen proved to be a very sensible tool to detect if a particular desmin mutation is able to cause filament abnormalities.

Variable pathogenic potentials of mutations located in the desmin alpha-helical domain.

Mutations in the desmin gene have been recognized as a cause of desminopathy, a familial or sporadic disorder characterized by skeletal muscle weakness, often associated with cardiomyopathy or respiratory insufficiency. Distinctive histopathologic features include aberrant intracytoplasmic accumulation of desmin (DES). We present here comparative phenotypic, molecular, and functional characteristics of four novel and three previously reported, but not fully characterized, desmin mutations localized in desmin alpha-helical domain. The results indicate that the c.638C>T (p.A213V), c.1178A>T (p.N393I), and to some extent the c.1078G>C (p.A360P) mutations exhibit pathogenic potentials only if combined with other mutations in desmin or other genes and should therefore be considered conditionally pathogenic. The c.1009G>C (p.A337P), c.1013T>G (p.L338R), c.1195G>T (p.D399Y), and c.1201G>A (p.E401K) mutations make desmin filaments dysfunctional and are capable of causing disease. The pathogenic potentials of desmin mutations correlate with the type and location of the disease-associated mutations in the relatively large and structurally and functionally complex desmin molecule. Mutations within the highly conserved alpha-helical structures are especially damaging since the integrity of the alpha-helix is critical for desmin filament assembly and stability.