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1.  Centronuclear Myopathy in Labrador Retrievers: A Recent Founder Mutation in the PTPLA Gene Has Rapidly Disseminated Worldwide 
PLoS ONE  2012;7(10):e46408.
Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ∼70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLAcnm mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLAcnm carriers in 13 countries. Haplotype analysis demonstrated that the PTPLAcnm allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM.
PMCID: PMC3465307  PMID: 23071563
2.  Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype–Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy 
Human mutation  2012;33(6):949-959.
Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype–phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot–Marie–Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT.
PMCID: PMC3374402  PMID: 22396310
centronuclear myopathy; congenital myopathy; Charcot–Marie–Tooth neuropathy; DNM2; ADCNM; CMTD1B; DI-CMTB; CMT2M; hereditary motor and sensory neuropathy type II; HMSNII; MTM1; myotubular myopathy; BIN1; RYR1; endocytosis
3.  Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy 
PLoS Genetics  2013;9(6):e1003430.
Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.
Author Summary
The intracellular organization of muscle fibers relies on a complex membrane system important for muscle structural organization, maintenance, contraction, and resistance to stress. Amphiphysin 2, encoded by BIN1, plays a central role in membrane sensing and remodelling and is involved in intracellular membrane trafficking in different cell types. The ubiquitously expressed BIN1, altered in centronuclear myopathy (CNM) and myotonic dystrophy (DM), possesses a muscle-specific exon coding for a phosphoinositide binding domain. We identified splice mutations affecting the muscle-specific BIN1 isoform in humans and dogs presenting a clinically and histopathologically comparable highly progressive centronuclear myopathy. Our functional and ultrastructural data emphasize the importance of amphiphysin 2 in membrane remodeling and suggest that the defective maintenance of the triad structure is a primary cause for the muscle weakness. The canine Inherited Myopathy of Great Danes is the first faithful mammalian model for investigating other potential pathological mechanisms underlying centronuclear myopathy and for testing therapeutic approaches.
PMCID: PMC3675003  PMID: 23754947
4.  Pathogenic Mechanisms in Centronuclear Myopathies 
Centronuclear myopathies (CNMs) are a genetically heterogeneous group of inherited neuromuscular disorders characterized by clinical features of a congenital myopathy and abundant central nuclei as the most prominent histopathological feature. The most common forms of congenital myopathies with central nuclei have been attributed to X-linked recessive mutations in the MTM1 gene encoding myotubularin (“X-linked myotubular myopathy”), autosomal-dominant mutations in the DNM2 gene encoding dynamin-2 and the BIN1 gene encoding amphiphysin-2 (also named bridging integrator-1, BIN1, or SH3P9), and autosomal-recessive mutations in BIN1, the RYR1 gene encoding the skeletal muscle ryanodine receptor, and the TTN gene encoding titin. Models to study and rescue the affected cellular pathways are now available in yeast, C. elegans, drosophila, zebrafish, mouse, and dog. Defects in membrane trafficking have emerged as a key pathogenic mechanisms, with aberrant T-tubule formation, abnormalities of triadic assembly, and disturbance of the excitation–contraction machinery the main downstream effects studied to date. Abnormal autophagy has recently been recognized as another important collateral of defective membrane trafficking in different genetic forms of CNM, suggesting an intriguing link to primary disorders of defective autophagy with overlapping histopathological features. The following review will provide an overview of clinical, histopathological, and genetic aspects of the CNMs in the context of the key pathogenic mechanism, outline unresolved questions, and indicate promising future lines of enquiry.
PMCID: PMC4271577  PMID: 25566070
centronuclear myopathy; myotubular myopathy; MTM1 myotubularin gene; DNM2 dynamin-2 gene; BIN1 bridging integrator-1/amphiphysin-2 gene; RYR1 ryanodine receptor-1 gene; TTN titin gene; autophagy
5.  Centronuclear (myotubular) myopathy 
Centronuclear myopathy (CNM) is an inherited neuromuscular disorder characterised by clinical features of a congenital myopathy and centrally placed nuclei on muscle biopsy.
The incidence of X-linked myotubular myopathy is estimated at 2/100000 male births but epidemiological data for other forms are not currently available.
The clinical picture is highly variable. The X-linked form usually gives rise to a severe phenotype in males presenting at birth with marked weakness and hypotonia, external ophthalmoplegia and respiratory failure. Signs of antenatal onset comprise reduced foetal movements, polyhydramnios and thinning of the ribs on chest radiographs; birth asphyxia may be the present. Affected infants are often macrosomic, with length above the 90th centile and large head circumference. Testes are frequently undescended. Both autosomal-recessive (AR) and autosomal-dominant (AD) forms differ from the X-linked form regarding age at onset, severity, clinical characteristics and prognosis. In general, AD forms have a later onset and milder course than the X-linked form, and the AR form is intermediate in both respects.
Mutations in the myotubularin (MTM1) gene on chromosome Xq28 have been identified in the majority of patients with the X-linked recessive form, whilst AD and AR forms have been associated with mutations in the dynamin 2 (DNM2) gene on chromosome 19p13.2 and the amphiphysin 2 (BIN1) gene on chromosome 2q14, respectively. Single cases with features of CNM have been associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the hJUMPY (MTMR14) genes.
Diagnosis is based on typical histopathological findings on muscle biopsy in combination with suggestive clinical features; muscle magnetic resonance imaging may complement clinical assessment and inform genetic testing in cases with equivocal features. Genetic counselling should be offered to all patients and families in whom a diagnosis of CNM has been made.
The main differential diagnoses include congenital myotonic dystrophy and other conditions with severe neonatal hypotonia.
Management of CNM is mainly supportive, based on a multidisciplinary approach. Whereas the X-linked form due to MTM1 mutations is often fatal in infancy, dominant forms due to DNM2 mutations and some cases of the recessive BIN1-related form appear to be associated with an overall more favourable prognosis.
PMCID: PMC2572588  PMID: 18817572
6.  Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy 
Neurology  2013;81(14):1205-1214.
To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes.
Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest.
Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations.
Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.
PMCID: PMC3795603  PMID: 23975875
7.  Expanding the MTM1 mutational spectrum: novel variants including the first multi-exonic duplication and development of a locus-specific database 
Myotubular myopathy (MIM#310400), the X-linked form of Centronuclear myopathy (CNM) is mainly characterized by neonatal hypotonia and inability to maintain unassisted respiration. The MTM1 gene, responsible for this disease, encodes myotubularin – a lipidic phosphatase involved in vesicle trafficking regulation and maturation. Recently, it was shown that myotubularin interacts with desmin, being a major regulator of intermediate filaments. We report the development of a locus-specific database for MTM1 using the Leiden Open Variation database software (, with data collated for 474 mutations identified in 472 patients (by June 2012). Among the entries are a total of 25 new mutations, including a large deletion encompassing introns 2–15. During database implementation it was noticed that no large duplications had been reported. We tested a group of eight uncharacterized CNM patients for this specific type of mutation, by multiple ligation-dependent probe amplification (MLPA) analysis. A large duplication spanning exons 1–5 was identified in a boy with a mild phenotype, with results pointing toward possible somatic mosaicism. Further characterization revealed that this duplication causes an in-frame deletion at the mRNA level (r.343_444del). Results obtained with a next generation sequencing approach suggested that the duplication extends into the neighboring MAMLD1 gene and subsequent cDNA analysis detected the presence of a MTM1/MAMLD1 fusion transcript. A complex rearrangement involving the duplication of exon 10 has since been reported, with detection also enabled by MLPA analysis. It is thus conceivable that large duplications in MTM1 may account for a number of CNM cases that have remained genetically unresolved.
PMCID: PMC3641378  PMID: 22968136
locus-specific database; MTM1; novel mutations
8.  Mild Functional Differences of Dynamin 2 Mutations Associated to Centronuclear Myopathy and Charcot-Marie-Tooth Peripheral Neuropathy 
PLoS ONE  2011;6(11):e27498.
The large GTPase dynamin 2 is a key player in membrane and cytoskeletal dynamics mutated in centronuclear myopathy (CNM) and Charcot-Marie Tooth (CMT) neuropathy, two discrete dominant neuromuscular disorders affecting skeletal muscle and peripheral nerves respectively. The molecular basis for the tissue-specific phenotypes observed and the physiopathological mechanisms linked to dynamin 2 mutations are not well established. In this study, we have analyzed the impact of CNM and CMT implicated dynamin 2 mutants using ectopic expression of four CNM and two CMT mutations, and patient fibroblasts harboring two dynamin 2 CNM mutations in established cellular processes of dynamin 2 action. Wild type and CMT mutants were seen in association with microtubules whereas CNM mutants lacked microtubules association and did not disrupt interphase microtubules dynamics. Most dynamin 2 mutants partially decreased clathrin-mediated endocytosis when ectopically expressed in cultured cells; however, experiments in patient fibroblasts suggested that endocytosis is overall not defective. Furthermore, CNM mutants were seen in association with enlarged clathrin stained structures whereas the CMT mutant constructs were associated with clathrin structures that appeared clustered, similar to the structures observed in Dnm1 and Dnm2 double knock-out cells. Other roles of dynamin 2 including its interaction with BIN1 (amphiphysin 2), and its function in Golgi maintenance and centrosome cohesion were not significantly altered. Taken together, these mild functional defects are suggestive of differences between CMT and CNM disease-causing dynamin 2 mutants and suggest that a slight impairment in clathrin-mediated pathways may accumulate over time to foster the respective human diseases.
PMCID: PMC3214065  PMID: 22096584
9.  Clinical and Pathological Features of Korean Patients with DNM2-Related Centronuclear Myopathy 
Background and Purpose
Centronuclear myopathy (CNM) is characterized by the presence of central nuclei within a large number of muscle fibers. Mutations of the dynamin 2 gene (DNM2) are common causes of autosomal dominant or sporadic CNM. The aim of this study was to characterize the clinical and pathological features of CNM relative to the presence of DNM2 mutations.
Six patients with clinical and pathological features of CNM were recruited. Detailed clinical and pathological findings were analyzed according to the presence of DNM2 mutations.
We detected DNM2 mutations in four of the six sporadic CNM patients, and identified the following distinct clinical and pathological features in those patients with DNM2 mutations: preferential involvement of the distal lower limbs, typical nuclear centralization, and radially distributed sarcoplasmic strands in muscle pathology. In contrast, those without DNM2 mutations exhibited rather diffuse muscular involvement, and nuclear internalization and myofibrillar disorganization were more pronounced features of their muscle pathology.
These findings suggest the presence of specific features in Korean CNM patients. A detailed clinical and pathological examination of CNM patients would be helpful for molecular genetic analyses of this condition.
PMCID: PMC3896645  PMID: 24465259
centronuclear myopathy; DNM2; muscle involvement; central nuclei; internal nuclei; sarcoplasmic strands
10.  Case report of intrafamilial variability in autosomal recessive centronuclear myopathy associated to a novel BIN1 stop mutation 
Centronuclear myopathies (CNM) describe a group of rare muscle diseases typically presenting an abnormal positioning of nuclei in muscle fibers. To date, three genes are known to be associated to a classical CNM phenotype. The X-linked neonatal form (XLCNM) is due to mutations in MTM1 and involves a severe and generalized muscle weakness at birth. The autosomal dominant form results from DNM2 mutations and has been described with early childhood and adult onset (ADCNM). Autosomal recessive centronuclear myopathy (ARCNM) is less characterized and has recently been associated to mutations in BIN1, encoding amphiphysin 2. Here we present the first clinical description of intrafamilal variability in two first-degree cousins with a novel BIN1 stop mutation. In addition to skeletal muscle defects, both patients have mild mental retardation and the more severely affected male also displays abnormal ventilation and cardiac arrhythmia, thus expanding the phenotypic spectrum of BIN1-related CNM to non skeletal muscle defects. We provide an up-to-date review of all previous cases with ARCNM and BIN1 mutations.
PMCID: PMC3014877  PMID: 21129173
11.  Reducing dynamin 2 expression rescues X-linked centronuclear myopathy 
The Journal of Clinical Investigation  2014;124(3):1350-1363.
Centronuclear myopathies (CNM) are congenital disorders associated with muscle weakness and abnormally located nuclei in skeletal muscle. An autosomal dominant form of CNM results from mutations in the gene encoding dynamin 2 (DNM2), and loss-of-function mutations in the gene encoding myotubularin (MTM1) result in X-linked CNM (XLCNM, also called myotubular myopathy), which promotes severe neonatal hypotonia and early death. Currently, no effective treatments exist for XLCNM. Here, we found increased DNM2 levels in XLCNM patients and a mouse model of XLCNM (Mtm1–/y). Generation of Mtm1–/y mice that were heterozygous for Dnm2 revealed that reduction of DNM2 in XLCNM mice restored life span, whole-body strength, and diaphragm function and increased muscle strength. Additionally, classic CNM-associated histological features, including fiber atrophy and nuclei mispositioning, were absent or reduced. Ultrastructural analysis revealed improvement of sarcomere organization and triad structures. Skeletal muscle–specific decrease of Dnm2 during embryogenesis or in young mice after disease onset revealed that the rescue associated with downregulation of Dnm2 is cell autonomous and is able to stop and potentially revert XLCNM progression. These data indicate that MTM1 and DNM2 regulate muscle organization and force through a common pathway. Furthermore, despite DNM2 being a key mechanoenzyme, its reduction is beneficial for XLCNM and represents a potential therapeutic approach for patients.
PMCID: PMC3938268  PMID: 24569376
12.  Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence 
Journal of Bacteriology  2014;196(15):2789-2797.
Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm+ strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.
PMCID: PMC4135665  PMID: 24837294
13.  Severe phenotype of a patient with autosomal recessive centronuclear myopathy due to a BIN1 mutation 
Acta Myologica  2009;28(3):91-93.
Centronuclear myopathy (CNM) is a rare hereditary congenital myopathy characterized by muscular hypotonia and abnormal centralization of nuclei in muscle fibers. The autosomal recessive (AR) form presents from birth to childhood, followed by a mild progression of muscle weakness. Despite recently identified genetic loci in the AR form, genotype-phenotype correlations are poorly established. Our index case is a 17 year old boy with recessive CNM causing loss of ambulation at 13 years of age and requiring ventilatory assistance nightly. Recent genetic testing revealed a c.1723A > T mutation in the BIN1 gene. The phenotype of the index case contrasts to previously published cases, where recessive CNM patients have lost ambulation in their 20s and have not required ventilatory assistance. The disease severity of our index case, carrying a c.1723A > T mutation, widens the phenotypic spectrum of AR CNM to include earlier loss of ambulation and respiratory failure.
PMCID: PMC2858945  PMID: 20476667
Centronuclear myopathy; BIN1; phenotype
14.  A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells 
Biochimica et biophysica acta  2013;1840(1):10.1016/j.bbagen.2013.09.001.
Dynamin 2 (Dyn2) is a ~100 kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause Centronuclear Myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state.
In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells.
Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1 μM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2.
Conclusions and General Significance
These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.
PMCID: PMC3859711  PMID: 24016602
Dynamin 2; Centronuclear myopathy; R369W mutation; EGFP; Fluorescence Fluctuation Spectroscopy; TIRF
15.  Sporadic Centronuclear Myopathy with Muscle Pseudohypertrophy, Neutropenia, and Necklace Fibers due to a DNM2 mutation 
Neuromuscular disorders : NMD  2010;20(12):801-804.
Dynamin 2 gene (DNM2) mutations result in an autosomal dominant centronuclear myopathy (CNM) and a Charcot-Marie-Tooth (CMT) neuropathy. DNM2-CMT but not DNM2-CNM patients were noted to have neutropenia. We here report a man with paravertebral muscles hypertrophy and mild neutropenia. His muscle biopsy was typical for CNM with additional “necklace” fibers. Sequencing of DNM2 revealed a known heterozygous c.1269C>T (p.Arg369Trp) mutation. Necklace fibers were considered as a pathological hallmark of late onset X-linked CNM due to mutations in MTM1 but have not been observed in DNM2-CNM. The findings broaden the features of DNM2-myopathy.
PMCID: PMC2991611  PMID: 20817456
Centronuclear myopathy; DNM2; dynamin 2; muscle hypertrophy; muscle pseudohypertrophy, necklace fibers; neutropenia
16.  Genetics of congenital nemaline myopathy: a study of 10 families. 
Journal of Medical Genetics  1990;27(8):480-487.
In order to investigate the inheritance in congenital nemaline myopathy (CNM), we studied the family histories and pedigrees of 13 patients with CNM from 10 families, and the 20 patients, by physical examination, single fibre electromyography, ultrasonography of muscles, measurement of serum creatine kinase, muscle biopsy, and electrophoresis of muscle proteins. None of the parents was affected. In three families there were two affected children. Of the parents, 15 showed deficiency of type 2B muscle fibres, and all except one father showed some other minor neuromuscular abnormality. These may represent heterozygous manifestations of recessive gene. Most of the ancestors came from sparsely populated rural communities in the west of Finland. We conclude that in the Finnish CNM patients, the mode of inheritance appears to be recessive. Apart from a few instances of dominant inheritance, most cases published also seem compatible with recessive inheritance.
PMCID: PMC1017195  PMID: 2213842
17.  X linked neonatal centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci. 
Journal of Medical Genetics  1990;27(5):284-287.
We have studied the inheritance of several polymorphic Xq27/28 DNA marker loci in two three generation families with the X linked neonatal lethal form of centronuclear/myotubular myopathy (XL MTM). We found complete linkage of XLMTM to all four informative Xq28 markers analysed, with GCP/RCP (Z = 3.876, theta = 0.00), with DXS15 (Z = 3.737, theta = 0.00), with DXS52 (Z = 2.709, theta = 0.00), and with F8C (Z = 1.020, theta = 0.00). In the absence of any observable recombination, we are unable to sublocalise the XLMTM locus further within the Xq28 region. This evidence for an Xq28 localisation may allow us to carry out useful genetic counselling within such families.
PMCID: PMC1017076  PMID: 2352256
18.  N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy 
EMBO Molecular Medicine  2014;6(11):1455-1475.
Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.
PMCID: PMC4237471  PMID: 25262827
centronuclear myopathy; cytoskeleton; nuclear movement; triad formation
19.  Common Membrane Trafficking Defects of Disease Associated Dynamin 2 Mutations 
Traffic (Copenhagen, Denmark)  2011;12(11):1620-1633.
Dynamin (Dyn) is a multidomain and multifunctional GTPase best known for its essential role in clathrin-mediated endocytosis (CME). Dyn2 mutations have been linked to two human diseases, Centronuclear Myopathy (CNM) and Charcot-Marie-Tooth (CMT) disease. Paradoxically, although Dyn2 is ubiquitously expressed and essential for embryonic development, the disease-associated Dyn2 mutants are autosomal dominant, but result in slowly progressing and tissue-specific diseases. Thus, although the cellular defects that cause disease remain unclear, they are expected to be mild. To gain new insight into potential pathogenic mechanisms we utilized mouse Dyn2 conditional knock-out cells combined with retroviral-mediated reconstitution to mimic both heterozygous and homozygous states and characterized cellular phenotypes using quantitative assays for several membrane trafficking events. Surprisingly, none of the four mutants studied exhibited a defect in CME, but all were impaired in their ability to support p75/neurotrophin receptor export from the Golgi, the raft-dependent endocytosis of cholera toxin, and clathrin-independent endocytosis of EGFR. While it will be important to study these mutants in disease-relevant muscle and neuronal cells, given the importance of neurotrophic factors and lipid rafts in muscle physiology, we speculate that these common cellular defects might contribute to the tissue-specific diseases caused by a ubiquitously expressed protein.
PMCID: PMC3188678  PMID: 21762456
Charcot-Marie-Tooth disease; Centronuclear Myopathy; Clathrin-mediated endocytosis; EGF receptor; p75/neurotrophin receptor; lipid rafts
20.  Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum: prevalences and investigations on a new transmission path in small mammals and ixodid ticks 
Parasites & Vectors  2014;7(1):563.
Small mammals are crucial for the life history of ixodid ticks, but their role and importance in the transmission cycle of tick-borne pathogens is mostly unknown. Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum are both tick-borne pathogens, and rodents are discussed to serve as main reservoir hosts for CNM but not for the latter especially in Germany. Analysing the prevalence of both pathogens in small mammals and their ticks in endemic regions may help to elucidate possible transmission paths in small mammal populations and between small mammals and ticks.
In 2012 and 2013, small mammals were trapped at three different sites in Germany. DNA was extracted from different small mammal tissues, from rodent neonates, foetuses and from questing and attached ticks. DNA samples were tested for CNM and A. phagocytophilum by real-time PCR. Samples positive for A. phagocytophilum were further characterized at the 16S rRNA gene locus.
CNM was detected in 28.6% of small mammals and in 2.2% of questing and 3.8% of attached ticks. Altogether 33 positive ticks were attached to 17 different hosts, while positive ticks per host ranged between one and seven. The prevalences for this pathogen differed significantly within small mammal populations comparing sites (χ2: 13.3987; p: 0.0004) and between sexes. Male rodents had an approximately two times higher chance of infection than females (OR: 1.9652; 95% CI: 1.32-2.92). The prevalence for CNM was 31.8% (95% CI: 22-44) in rodent foetuses and neonates (23 of 67) from positive dams, and 60% (95% CI: 35.7-80.25) of positive gravid or recently parturient rodents (9 out of 15) had at least one positive foetus or neonate. Anaplasma phagocytophilum was detected at a low percentage in rodents (0-5.6%) and host-attached ticks (0.5-2.9%) with no significant differences between rodent species. However, attached nymphs were significantly more often infected than attached larvae (χ2: 25.091; p: <0.0001).
This study suggests that CNM is mainly a rodent-associated pathogen and provides evidence for a potential transplacental transmission in rodents. In contrast, most of the rodent species captured likely represent only accidental hosts for A. phagocytophilum at the investigated sites.
PMCID: PMC4264555  PMID: 25465390
Candidatus Neoehrlichia mikurensis; Anaplasma phagocytophilum; Ixodes ricinus; Emerging pathogen; Small mammals; Tick-borne Pathogen
21.  The Collagen-Binding Protein Cnm Is Required for Streptococcus mutans Adherence to and Intracellular Invasion of Human Coronary Artery Endothelial Cells ▿  
Infection and Immunity  2011;79(6):2277-2284.
Streptococcus mutans is considered the primary etiologic agent of dental caries, a global health problem that affects 60 to 90% of the population, and a leading causative agent of infective endocarditis. It can be divided into four different serotypes (c, e, f, and k), with serotype c strains being the most common in the oral cavity. In this study, we demonstrate that in addition to OMZ175 and B14, three other strains (NCTC11060, LM7, and OM50E) of the less prevalent serotypes e and f are able to invade primary human coronary artery endothelial cells (HCAEC). Invasive strains were also significantly more virulent than noninvasive strains in the Galleria mellonella (greater wax worm) model of systemic disease. Interestingly, the invasive strains carried an additional gene, cnm, which was previously shown to bind to collagen and laminin in vitro. Inactivation of cnm rendered the organisms unable to invade HCAEC and attenuated their virulence in G. mellonella. Notably, the cnm knockout strains did not adhere to HCAEC as efficiently as the parental strains did, indicating that the loss of the invasion phenotype observed for the mutants was linked to an adhesion defect. Comparisons of the invasive strains and their respective cnm mutants did not support a correlation between biofilm formation and invasion. Thus, Cnm is required for S. mutans invasion of endothelial cells and possibly represents an important virulence factor of S. mutans that may contribute to cardiovascular infections and pathologies.
PMCID: PMC3125845  PMID: 21422186
22.  Dynamin 2 the rescue for centronuclear myopathy 
Centronuclear myopathy is a lethal muscle disease. The most severe form of the disease, X-linked centronuclear myopathy, is due to mutations in the gene encoding myotubularin (MTM1), while mutations in dynamin 2 (DNM2) and amphiphysin 2/BIN1 (AMPH2) cause milder forms of myopathy. MTM1 is a lipid phosphatase, and mutations that disrupt this activity cause severe muscle wasting. In this issue of the JCI, Cowling and colleagues report on their finding of increased DNM2 levels in human and mouse muscle with MTM1 mutations. Partial reduction of Dnm2 in mice harboring Mtm1 mutations remarkably rescued muscle wasting and lethality, and this effect was muscle specific. DNM2 regulates membrane trafficking through vesicular scission, and it is presumed that reducing this activity accounts for improved outcome in X-linked centronuclear myopathy.
PMCID: PMC3938257  PMID: 24569368
23.  Phylogeny of Penicillium and the segregation of Trichocomaceae into three families 
Studies in Mycology  2011;70(1):1-51.
Species of Trichocomaceae occur commonly and are important to both industry and medicine. They are associated with food spoilage and mycotoxin production and can occur in the indoor environment, causing health hazards by the formation of β-glucans, mycotoxins and surface proteins. Some species are opportunistic pathogens, while others are exploited in biotechnology for the production of enzymes, antibiotics and other products. Penicillium belongs phylogenetically to Trichocomaceae and more than 250 species are currently accepted in this genus. In this study, we investigated the relationship of Penicillium to other genera of Trichocomaceae and studied in detail the phylogeny of the genus itself. In order to study these relationships, partial RPB1, RPB2 (RNA polymerase II genes), Tsr1 (putative ribosome biogenesis protein) and Cct8 (putative chaperonin complex component TCP-1) gene sequences were obtained. The Trichocomaceae are divided in three separate families: Aspergillaceae, Thermoascaceae and Trichocomaceae. The Aspergillaceae are characterised by the formation flask-shaped or cylindrical phialides, asci produced inside cleistothecia or surrounded by Hülle cells and mainly ascospores with a furrow or slit, while the Trichocomaceae are defined by the formation of lanceolate phialides, asci borne within a tuft or layer of loose hyphae and ascospores lacking a slit. Thermoascus and Paecilomyces, both members of Thermoascaceae, also form ascospores lacking a furrow or slit, but are differentiated from Trichocomaceae by the production of asci from croziers and their thermotolerant or thermophilic nature. Phylogenetic analysis shows that Penicillium is polyphyletic. The genus is re-defined and a monophyletic genus for both anamorphs and teleomorphs is created (Penicillium sensu stricto). The genera Thysanophora, Eupenicillium, Chromocleista, Hemicarpenteles and Torulomyces belong in Penicillium s. str. and new combinations for the species belonging to these genera are proposed. Analysis of Penicillium below genus rank revealed the presence of 25 clades. A new classification system including both anamorph and teleomorph species is proposed and these 25 clades are treated here as sections. An overview of species belonging to each section is presented.
Taxonomic novelties:
New sections, all in Penicillium: sect. Sclerotiora Houbraken & Samson, sect. Charlesia Houbraken & Samson, sect. Thysanophora Houbraken & Samson,sect. Ochrosalmonea Houbraken & Samson, sect. Cinnamopurpurea Houbraken & Samson, Fracta Houbraken & Samson, sect. Stolkia Houbraken & Samson, sect. Gracilenta Houbraken & Samson, sect. Citrina Houbraken & Samson, sect. Turbata Houbraken & Samson, sect. Paradoxa Houbraken & Samson, sect. Canescentia Houbraken & Samson.
New combinations: Penicillium asymmetricum (Subramanian & Sudha) Houbraken & Samson, P. bovifimosum (Tuthill & Frisvad) Houbraken & Samson, P. glaucoalbidum (Desmazières) Houbraken & Samson, P. laeve (K. Ando & Manoch) Houbraken & Samson, P. longisporum (Kendrick) Houbraken & Samson, P. malachiteum (Yaguchi & Udagawa) Houbraken & Samson, P. ovatum (K. Ando & Nawawi) Houbraken & Samson, P. parviverrucosum (K. Ando & Pitt) Houbraken & Samson, P. saturniforme (Wang & Zhuang) Houbraken & Samson, P. taiwanense (Matsushima) Houbraken & Samson.
New names: Penicillium coniferophilum Houbraken & Samson, P. hennebertii Houbraken & Samson, P. melanostipe Houbraken & Samson, P. porphyreum Houbraken & Samson.
PMCID: PMC3233907  PMID: 22308045
Aspergillus; Eupenicillium; nomenclature; Penicillium; Talaromyces; taxonomy.
24.  Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization 
PLoS ONE  2014;9(4):e93060.
The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.
PMCID: PMC3995651  PMID: 24755653
25.  Clinical and molecular genetic characterisation of a family segregating autosomal dominant retinitis pigmentosa and sensorineural deafness 
AIMS/BACKGROUND—To characterise clinically a large kindred segregating retinitis pigmentosa and sensorineural hearing impairment in an autosomal dominant pattern and perform genetic linkage studies in this family. Extensive linkage analysis in this family had previously excluded the majority of loci shown to be involved in the aetiologies of RP, some other forms of inherited retinal degeneration, and inherited deafness.
METHODS—Members of the family were subjected to detailed ophthalmic and audiological assessment. In addition, some family members underwent skeletal muscle biopsy, electromyography, and electrocardiography. Linkage analysis using anonymous microsatellite markers was performed on DNA samples from all living members of the pedigree.
RESULTS—Patients in this kindred have a retinopathy typical of retinitis pigmentosa in addition to a hearing impairment. Those members of the pedigree examined demonstrated a subclinical myopathy, as evidenced by abnormal skeletal muscle histology, electromyography, and electrocardiography. LOD scores of Zmax = 3. 75 (Θ = 0. 10), Zmax = 3. 41 (Θ = 0. 10), and Zmax = 3. 25 (Θ = 0. 15) respectively were obtained with the markers D9S118, D9S121, and ASS, located on chromosome 9q34-qter, suggesting that the causative gene in this family may lie on the long arm (q) of chromosome 9.
CONCLUSIONS—These data indicate that the gene responsible for the phenotype in this kindred is located on chromosome 9q. These data, together with evidence that a murine deafness gene is located in a syntenic area of the mouse genome, should direct the research community to consider this area as a candidate region for retinopathy and/or deafness genes.

PMCID: PMC1722127  PMID: 9135384

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