The influence of glycosaminoglycan proteoglycan side chains on tensile force transmission and the nanostructural properties of Achilles tendons.

This study investigates the nanostructural mechanisms that lie behind load transmission in tendons and the role of glycosaminoglycans (GAGs) in the transmission of force in the tendon extracellular matrix. The GAGs in white New Zealand rabbit Achilles tendons were enzymatically depleted, and the tendons subjected to cyclic loading at 6% strain for up to 2 hr. A nanoscale morphometric assessment of fibril deformation under strain was linked with the decline in the tendon macroscale mechanical properties. An atomic force microscope (AFM) was employed to characterize the D-periodicity within and between fibril bundles (WFB and BFB, respectively). By the end of the second hour of the applied strain, the WFB and BFB D-periodicities had significantly increased in the GAG-depleted group (29% increase compared with 15% for the control, p < .0001). No statistically significant differences were found between WFB and BFB D-periodicities in either the control or GAG-depleted groups, suggesting that mechanical load in Achilles tendons is uniformly distributed and fairly homogenous among the WFB and BFB networks. The results of this study have provided evidence of a cycle-dependent mechanism of damage accumulation. The accurate quantification of fibril elongation (measured as the WFB and BFB D-periodicity lengths) in response to macroscopic applied strain has assisted in assessing the complex structure-function relationship in Achilles tendon.

Contribution of glycosaminoglycans to the structural and mechanical properties of tendons - A multiscale study.

Tendinopathy of the Achilles tendon contributes to a large range of disorders, including mechanical damage and degenerative diseases. Glycosaminoglycans (GAGs), are thought to play a role in the mechanical strength of tendons by forming cross-links between collagen molecules and allowing the transmission of forces between fibrils. This study assessed the response of GAG-depleted tendons to damage induced by fatigue loading, investigating the mechanical damage (stiffness, hysteresis and maximum load), macrostructural changes (tenocyte morphology, fiber anisotropy and waviness) assessed by confocal imaging and nanostructural changes (fibril D-periodicity length) within the same non-viable intact tendons. Changes in fiber waviness and tenocyte shape are strongly correlated to mechanical and nano-structural (D-periodicity elongation) properties in both Control and GAG-depleted tendons. This study supports firstly, the vital role GAGs play as mechanical connectors facilitating the load transfer between the fibrils and their hydrophilic role in facilitating fibril sliding. Secondly, that observed changes in tenocyte shape and fiber waviness correlate with tendon stiffness and other mechanical profiles.

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics.

The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell's fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

CD8+ cytotoxic T cell responses to dominant tumor-associated antigens are profoundly weakened by aging yet subdominant responses retain functionality and expand in response to chemotherapy.

Increasing life expectancy is associated with increased cancer incidence, yet the effect of cancer and anti-cancer treatment on elderly patients and their immune systems is not well understood. Declining T cell function with aging in response to infection and vaccination is well documented, however little is known about aged T cell responses to tumor antigens during cancer progression or how these responses are modulated by standard chemotherapy. We examined T cell responses to cancer in aged mice using AE17sOVA mesothelioma in which ovalbumin (OVA) becomes a 'spy' tumor antigen containing one dominant (SIINFEKL) and two subdominant (KVVRFDKL and NAIVFKGL) epitopes. Faster progressing tumors in elderly (22-24 months, cf. 60-70 human years) relative to young (2-3 months, human 15-18 years) mice were associated with increased pro-inflammatory cytokines and worsened cancer cachexia. Pentamer staining and an in-vivo cytotoxic T lymphocyte (CTL) assay showed that whilst elderly mice generated a greater number of CD8+ T cells recognizing all epitopes, they exhibited a profound loss of function in their ability to lyse targets expressing the dominant, but not subdominant, epitopes compared to young mice. Chemotherapy was less effective and more toxic in elderly mice however, similar to young mice, chemotherapy expanded CTLs recognizing at least one subdominant epitope in tumors and draining lymph nodes, yet treatment efficacy still required CD8+ T cells. Given the significant dysfunction associated with elderly CTLs recognizing dominant epitopes, our data suggest that responses to subdominant tumor epitopes may become important when elderly hosts with cancer are treated with chemotherapy.

Interaction Between Skeletal Muscle Cells and Extracellular Matrix Proteins Using a Serum Free Culture System.

The ability to grow C2C12 myoblasts in a completely defined, serum free medium enables researchers to investigate the role of specific factors in myoblast proliferation, migration, fusion, and differentiation without the confounding effects of serum. The use of defined, animal free in vitro culture systems will improve reproducibility between research groups and may also enhance translation of tissue engineering techniques into clinical applications. Here, we describe the use and characterization of a serum free culture system for C2C12 myoblasts using standard tissue culture medium and readily available, defined growth factors and supplements.

Macrophage Depletion in Elderly Mice Improves Response to Tumor Immunotherapy, Increases Anti-tumor T Cell Activity and Reduces Treatment-Induced Cachexia.

Most cancers emerge in the elderly, including lung cancer and mesothelioma, yet the elderly remain an underrepresented population in pre-clinical cancer studies and clinical trials. The immune system plays a critical role in the effectiveness of many anti-cancer therapies in young hosts via tumor-specific T cells. However, immunosuppressive macrophages can constitute up to 50% of the tumor burden and impair anti-tumor T cell activity. Altered macrophage phenotype and function during aging may further impact anti-tumor T cell responses. Yet, the impact of macrophages on anti-tumor T cell responses and immunotherapy in the elderly is unknown. Therefore, we examined macrophages and their interaction with T cells in young (3 months) and elderly (20-24 months) AE17 mesothelioma-bearing female C57BL/6J mice during tumor growth. Mesothelioma tumors grew faster in elderly compared with young mice, and this corresponded with an increase in tumor-associated macrophages. During healthy aging, macrophages increase in bone marrow and spleens suggesting that these sites have an increased potential to supply cancer-promoting macrophages. Interestingly, in tumor-bearing mice, bone marrow macrophages increased proliferation whilst splenic macrophages had reduced proliferation in elderly compared with young mice, and macrophage depletion using the F4/80 antibody slowed tumor growth in young and elderly mice. We also examined responses to treatment with intra-tumoral IL-2/anti-CD40 antibody immunotherapy and found it was less effective in elderly (38% tumor regression) compared to young mice (90% regression). Tumor-bearing elderly mice decreased in vivo anti-tumor cytotoxic T cell activity in tumor draining lymph nodes and spleens. Depletion of macrophages using F4/80 antibody in elderly, but not young mice, improved IL-2/anti-CD40 immunotherapy up to 78% tumor regression. Macrophage depletion also increased in vivo anti-tumor T cell activity in elderly, but not young mice. All the tumor-bearing elderly (but not young) mice had decreased body weight (i.e., exhibited cachexia), which was greatly exacerbated by immunotherapy; whereas macrophage depletion prevented this immunotherapy-induced cachexia. These studies strongly indicate that age-related changes in macrophages play a key role in driving cancer cachexia in the elderly, particularly during immunotherapy, and sabotage elderly anti-tumor immune responses.

WITHDRAWN: The Fundamental And Pathological Importance Of Oxysterol Binding Protein And Its Related Proteins.

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Aged neutrophils accumulate in lymphoid tissues from healthy elderly mice and infiltrate T- and B-cell zones.

The average age of the human population is rising, leading to an increasing burden of age-related diseases, including increased susceptibility to infection. However, immune function can decrease with age which could impact on processes that require a functional immune system. Aging is also characterized by chronic low-grade inflammation which could further impact immune cell function. While changes to neutrophils in blood during aging have been described, little is known in aging lymphoid organs. This study used female C57BL/6J mice comparing bone marrow (BM), spleen and lymph nodes from young mice aged 2-3 months (equivalent to 18 human years) with healthy elderly mice aged 22-24 months (equivalent to 60-70 human years). Neutrophil proportions increased in BM and secondary lymphoid organs of elderly mice relative to their younger counterparts and presented an atypical phenotype. Interestingly, neutrophils from elderly spleen and lymph nodes were long lived (with decreased apoptosis via Annexin V staining and increased proportion of BrdUneg mature cells) with splenic neutrophils also demonstrating a hypersegmented morphology. Furthermore, splenic neutrophils of elderly mice expressed a mixed phenotype with increased expression of activation markers, CD11b and ICAM-1, increased proinflammatory TNFα, yet increased anti-inflammatory transforming growth factor-beta. Elderly splenic architecture was compromised, as the marginal zone (required for clearing infections) was contracted. Moreover, neutrophils from elderly but not young mice accumulated in lymph node and splenic T- and B-cell zones. Overall, the expansion of functionally compromised neutrophils could contribute to increased susceptibility to infection observed in the elderly.

Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

Lipid-laden partially-activated plasmacytoid and CD4(-)CD8α(+) dendritic cells accumulate in tissues in elderly mice.

BACKGROUND: Aging is associated with a decline in lymphocyte function however, little is known about dendritic cell (DC) subsets and aging. Aging is also associated with increasing circulating lipid levels and intracellular lipid accumulation modulates DC function. Whether age-associated increases in lipid levels influence DC biology is unknown. Thus, the effects of aging on DC subsets were assessed in vivo using young adult and elderly C57BL/6 J mice. RESULTS: Major age-related changes included increased CD11c(+) DC numbers in lymph nodes, spleens and livers, but not lungs, and significantly increased proportions of plasmacytoid (pDC) and CD4(-)CD8α(+) DCs in lymph nodes and livers. Other changes included altered pDC activation status (decreased CD40, increased MHC class-I and MHC class-II), increased lipid content in pDCs and CD4(-)CD8α(+) DCs, and increased expression of key mediators of lipid uptake including lipoprotein lipase, scavenger receptors (CD36, CD68 and LRP-1) in most tissues. CONCLUSIONS: Aging is associated with organ-specific numerical changes in DC subsets, and DC activation status, and increased lipid content in pDCs and CD4(-)CD8α(+) DCs. Up-regulation of lipoprotein lipase and scavenger receptors by lipid-rich pDCs and CD4(-)CD8α(+) DCs suggests these molecules contribute to DC lipid accumulation in the elderly. Lipid accumulation and modulated activation in pDCs and CD4(-)CD8α(+) DCs may contribute to the declining responses to vaccination and infection with age.

The Western Australian family connections genealogical project: detection of familial occurrences of single gene and chromosomal disorders.

AIM: To investigate using a Western Australian (WA) genealogical database for the identification of single gene and chromosome disorders among families. METHOD: Hospital admissions for single gene and chromosome disorders recorded during 2000-2006 were identified from the WA Hospital Morbidity Data System. The proportion of these conditions occurring in family groups was then identified using genealogical links created through the WA Family Connections Genealogical Project. RESULTS: There were 216 family clusters among 11,303 people who were recorded as having a genetic or chromosomal disorder on their hospital admission record. The most common single gene conditions found to occur in multiple family members included blood clotting disorders such as Factor VIII deficiency and Von Willebrand's disease, followed by cystic fibrosis, myotonic dystrophies, neurofibromatosis, tuberous sclerosis, and osteogenesis imperfecta. DISCUSSION: Single gene disorders most commonly occurring in multiple family members have been identified using the WA Family Connections Genealogical Project. These disorders reflect the most common single gene disorders requiring hospital admission, but which are not fatal before reproductive age and do not result in a loss of fertility. They are also restricted to disorders with earlier onset, as the WA Family Connections Genealogical Project currently covers 2-3 of the most recent generations. This study demonstrates the utility of record linkage genealogies to identify kindred with genetic disorders, offering a rich resource of information for focused genetic epidemiological research.

Melanoma biomolecules: independently identified but functionally intertwined.

The majority of patients diagnosed with melanoma present with thin lesions and generally these patients have a good prognosis. However, 5% of patients with early melanoma (<1 mm thick) will have recurrence and die within 10 years, despite no evidence of local or metastatic spread at the time of diagnosis. Thus, there is a need for additional prognostic markers to help identify those patients that may be at risk of recurrent disease. Many studies and several meta-analyses have compared gene and protein expression in melanocytes, naevi, primary, and metastatic melanoma in an attempt to find informative prognostic markers for these patients. However, although a large number of putative biomarkers have been described, few of these molecules are informative when used in isolation. The best approach is likely to involve a combination of molecules. We believe one approach could be to analyze the expression of a group of interacting proteins that regulate different aspects of the metastatic pathway. This is because a primary lesion expressing proteins involved in multiple stages of metastasis may be more likely to lead to secondary disease than one that does not. This review focuses on five putative biomarkers - melanoma cell adhesion molecule (MCAM), galectin-3 (gal-3), matrix metalloproteinase 2 (MMP-2), chondroitin sulfate proteoglycan 4 (CSPG4), and paired box 3 (PAX3). The goal is to provide context around what is known about the contribution of these biomarkers to melanoma biology and metastasis. Although each of these molecules have been independently identified as likely biomarkers, it is clear from our analyses that each are closely linked with each other, with intertwined roles in melanoma biology.

The role of immunoglobulin superfamily cell adhesion molecules in cancer metastasis.

Metastasis is a major clinical problem and results in a poor prognosis for most cancers. The metastatic pathway describes the process by which cancer cells give rise to a metastatic lesion in a new tissue or organ. It consists of interconnecting steps all of which must be successfully completed to result in a metastasis. Cell-cell adhesion is a key aspect of many of these steps. Adhesion molecules belonging to the immunoglobulin superfamily (Ig-SF) commonly play a central role in cell-cell adhesion, and a number of these molecules have been associated with cancer progression and a metastatic phenotype. Surprisingly, the contribution of Ig-SF members to metastasis has not received the attention afforded other cell adhesion molecules (CAMs) such as the integrins. Here we examine the steps in the metastatic pathway focusing on how the Ig-SF members, melanoma cell adhesion molecule (MCAM), L1CAM, neural CAM (NCAM), leukocyte CAM (ALCAM), intercellular CAM-1 (ICAM-1) and platelet endothelial CAM-1 (PECAM-1) could play a role. Although much remains to be understood, this review aims to raise the profile of Ig-SF members in metastasis formation and prompt further research that could lead to useful clinical outcomes.

The impact of single gene and chromosomal disorders on hospital admissions in an adult population.

Although the role of single gene and chromosomal disorders in pediatric illness has been recognized since the 1970s, there are few data describing the impact of these often severe disorders on the health of the adult population. In this study, we present population data describing the impact of single gene and chromosomal disorders on hospital admissions of patients aged 20 years and over in Western Australia between 2000 and 2006. The number, length, and cost of admissions were investigated and compared between disease categories and age groups and to hospital admissions for any reason. In total, 73,211 admissions and 8,032 patients were included in the study. The most costly disorders were cystic kidney disease, α-1 anti-trypsin deficiency, hemochromatosis, von Willebrand disease, and cystic fibrosis. Overall, patients with single gene and chromosomal disorders represented 0.5% of the patient population and were responsible for 1.9% of admissions and 1.5% of hospital costs. These data will enable informed provision of health care services for adults with single gene and chromosomal disorders in Australia.

Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1).

The ACTA1 gene encodes skeletal muscle alpha-actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease-causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease-causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core-like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype-phenotype correlations, we have developed a locus-specific database for ACTA1 variations (http://waimr.uwa.edu.au).

Mutations in TPM3 are a common cause of congenital fiber type disproportion.

OBJECTIVE: Congenital fiber type disproportion (CFTD) is a rare form of congenital myopathy in which the principal histological abnormality is hypotrophy of type 1 (slow-twitch) fibers compared with type 2 (fast-twitch) fibers. To date, mutation of ACTA1 and SEPN1 has been associated with CFTD, but the genetic basis in most patients is unclear. The gene encoding alpha-tropomyosin(slow) (TPM3) is a rare cause of nemaline myopathy, previously reported in only five families. We investigated whether mutation of TPM3 is a cause of CFTD. METHODS AND RESULTS: We sequenced TPM3 in 23 unrelated probands with CFTD or CFTD-like presentations of unknown cause and identified novel heterozygous missense mutations in five CFTD families (p. Leu100Met, p.Arg168Cys, p.Arg168Gly, p.Lys169Glu, p.Arg245Gly). All affected family members that underwent biopsy had typical histological features of CFTD, with type 1 fibers, on average, at least 50% smaller than type 2 fibers. We also report a sixth family in which a recurrent TPM3 mutation (p.Arg168His) was associated with histological features of CFTD and nemaline myopathy in different family members. We describe the clinical features of 11 affected patients. Typically, there was proximal limb girdle weakness, prominent weakness of neck flexion and ankle dorsiflexion, mild facial weakness, and mild ptosis. The age of onset and severity varied, even within the same family. Many patients required nocturnal noninvasive ventilation despite remaining ambulant. INTERPRETATION: Mutation of TPM3 is the most common cause of CFTD reported to date.

Myosin storage (hyaline body) myopathy: a case report.

Myosin storage myopathy/hyaline body myopathy is a rare congenital myopathy, with less than 30 cases reported in the literature. It is characterised by the presence of subsarcolemmal hyaline bodies in type 1 muscle fibres and predominantly proximal muscle weakness. Recently, a single mutation (Arg1845Trp) in the slow/beta-cardiac myosin heavy chain gene (MYH7) was identified in four unrelated probands from Sweden and Belgium. The clinical severity and age of onset was variable, despite the same disease-causing mutation and similar histological findings. Here, we report the clinical and morphological findings of two brothers of English/Scottish background with the Arg1845Trp mutation in MYH7. This case report adds to the clinical description of this rare disorder and confirms that Arg1845Trp is a common mutation associated with this phenotype, at least in the White European population.

Novel slow-skeletal myosin (MYH7) mutation in the original myosin storage myopathy kindred.

Myosin storage myopathy (OMIM 608358), a congenital myopathy characterised by subsarcolemmal, hyaline-like accumulations of myosin in Type I muscle fibres, was first described by Cancilla and Colleagues in 1971 [Neurology 1971;21:579-585] in two siblings as 'familial myopathy with probable lysis of myofibrils in type I muscle fibres'. Two mutations in the slow skeletal myosin heavy chain gene (MYH7) have recently been associated with the disease in other families. We have identified a novel heterozygous Leu1793Pro mutation in MYH7 in DNA from paraffin sections of one of the original siblings. This historical molecular analysis confirms the original cases had myosin storage myopathy.

Actin mutations are one cause of congenital fibre type disproportion.

We report three heterozygous missense mutations of the skeletal muscle alpha actin gene (ACTA1) in three unrelated cases of congenital fiber type disproportion (CFTD) from Japan and Australia. This represents the first genetic cause of CFTD to be identified and confirms that CFTD is genetically heterogeneous. The three mutations we have identified Leucine221Proline, Aspartate292Valine, and Proline332Serine are novel. They have not been found previously in any cases of nemaline, actin, intranuclear rod, or rod-core myopathy caused by mutations in ACTA1. It remains unclear why these mutations cause type 1 fiber hypotrophy but no nemaline bodies. The three mutations all lie on one face of the actin monomer on the surface swept by tropomyosin during muscle activity, which may suggest a common pathological mechanism. All three CFTD cases with ACTA1 mutations had severe congenital weakness and respiratory failure without ophthalmoplegia. There were no clinical features specific to CFTD cases with ACTA1 mutations, but the presence of normal eye movements in a severe CFTD patient may be an important clue for the presence of a mutation in ACTA1.