Dominant Myopathy Research Articles

Laing distal myopathy pathologically resembling inclusion body myositis.

Mutations in MYH7 cause autosomal dominant Laing distal myopathy. We present a family with a previously reported deletion (c.5186_5188delAGA, p.K1729del). Muscle pathology in one family member was characterized by an inflammatory myopathy with rimmed vacuoles, increased MHC Class I expression, and perivascular and endomysial muscle inflammation comprising CD3+, CD4+, CD8+, and CD68+ inflammatory cells. Interestingly, this biopsy specimen contained TDP-43, p62, and SMI-31-positive protein aggregates typical of inclusion body myositis. These findings should alert physicians to the possibility that patients with MYH7 mutations may have muscle biopsies showing pathologic findings similar to inclusion body myositis.

Adult-onset autosomal dominant centronuclear myopathy due to BIN1 mutations.

Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.

SKIN-DEEP; CLUES TO THE CAUSE OF PROXIMAL MYOPATHY

A 50 year old gentleman re-presented to a neurology clinic with a 16 year history of upper limb girdle proximal weakness, progressively weak in his proximal lower limbs and with...

Mutation in the novel nuclear-encoded mitochondrial protein CHCHD10 in a family with autosomal dominant mitochondrial myopathy.

Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5% of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

A.P.5: Novel recessive myotilin mutation cause severe myofibrillar myopathy

We identified the first homozygous and hence recessive mutation in the myotilin gene (MYOT) in a family affected by a severe myofibrillar myopathy (MFM). MFM is a rare, progressive, and devastating disease of human skeletal muscle with distinct histopathological pattern of protein aggregates and myofibrillar degeneration. So far, only heterozygous missense mutations in MYOT have been associated with autosomal dominant myofibrillar myopathy, limb girdle muscular dystrophy type 1A, and distal myopathy. Myotilin itself is highly expressed in skeletal and cardiac muscle, and is localized at the Z-disc and therefore interacts in sarcomere assembly. We performed whole-exome sequencing in a German family clinically diagnosed with MFM and identified a homozygous mutation in exon 2, c.16C>G (p.Arg6Gly). Using laser microdissection followed by quantitative mass spectrometry we identified the myotilin protein as one component showing highest increased abundance in the aggregates in the index patient. We suggest that the combined approach has a high potential as a new tool for the confirmation of unclassified variants which are found in whole-exome sequencing approaches.

G.P.45: Therapy of the dynamin 2-linked centronuclear myopathy by mRNA repair using Spliceosome-Mediated RNA Trans-splicing (SMarT) strategy

The autosomal dominant centronuclear myopathy (AD-CNM) is a rare congenital myopathy defined by skeletal muscle weakness and characteristic histopathological changes. Heterozygous mutations in the DNM2 gene are associated with entire clinical spectrum of AD-CNM. DNM2 gene encodes dynamin 2 (DNM2), a large GTPase ubiquitously expressed and involved in membrane trafficking. A Knock-In mouse model (KI-Dnm2R465W) expressing the most frequent mutation found in patients has been recently developed in the laboratory. The heterozygous mice progressively develop a muscle phenotype which recapitulates many aspects of the human condition. The purpose of this project is to evaluate the therapeutic potential of DNM2-mRNA repair by Spliceosome-Mediated RNA Trans-splicing (SMarT) technology able to reprogram the 5′, 3′ or internal coding sequence of endogenous mRNA. We have shown that classical 3′ strategy cannot be considered as accurate therapeutic strategy regarding the toxic effect of the Pre-Transplicing Molecules (PTMs) leading to low rate of trans-splicing in vivo. Thus, we tested several alternative strategies in order to prevent this toxicity and enhance frequency of trans-splicing events. We finally succeeded to overcome the toxicity using a 5′ trans-splicing strategy and obtained hopeful results. Indeed, we detected trans-splicing events at mRNA and protein levels in vitro and in vivo.

A.P.9: Elucidation of the mechanism of disease in BAG3-related myofibrillar myopathy

Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been shown to cause myofibrillar myopathy (MFM). MFM is characterised at the cellular level by the formation of protein aggregates and structural failure of the Z-disk. In contrast to the other MFM causing proteins BAG3 has no structural role in the Z-disk but is involved in the regulation of autophagy and the degradation of misfolded proteins. Protein aggregation in BAG3-related MFM has therefore been proposed to result from a reduction in autophagic activity. To investigate the mechanism of disease in BAG3-related MFM we expressed the human wildtype BAG3 or the dominant myofibrillar myopathy causing mutant form BAG3^P209L in zebrafish muscle. The expression of BAG3^P209L resulted in protein aggregation and we performed time-lapse imaging to examine aggregate formation in vivo and examined their composition. To investigate the role of impaired autophagy in BAG related MFM we stimulated or inhibited autophagy in BAG3^wt or BAG3^P209L expressing embryos and examined its effect on protein aggregation. Our studies revealed that autophagy is active in BAG3^P209L expressing fish and that inhibition of autophagy is not sufficient to induce protein aggregation, suggesting that the pathology is not due to impaired autophagic activity. We did not observe fibre failure in the BAG3^P209L model fish and therefore examined the effect of knocking down BAG3 expression. Loss of BAG3 function resulted in fibre failure but not the formation of protein aggregates. Together our knockdown and overexpressing models demonstrate contraction-dependent fiber failure and the formation of protein aggregates. Furthermore, our experimental analysis of the transgenic and knockdown embryos demonstrates that protein aggregation is not due to an inhibition of autophagy and suggests a novel mechanism of disease with the cellular pathology caused by a combination of toxic gain of function and BAG3 insufficiency.

G.P.56: A de novo mutation of the MYH7 gene in a large Chinese family with autosomal dominant myopathy

Laing distal myopathy (LDM) is an autosomal dominant myopathy which is caused by mutation in the slow/beta cardiac myosin heavy chain (<i>MYH7</i>) gene. It has been recently reported that LDM presents with a wide range of clinical manifestations. We herein report a large Chinese family with autosomal dominant myopathy. The affected individuals in the family presented foot drop at early childhood, and progressive distal and proximal limb weakness. Their characteristic symptoms were scapular winging and scoliosis in early disease phase, and impairment of ambulation in advanced phase. At first, we had suspected limb girdle muscular dystrophy (LGMD), but had not reached a correct diagnosis. Next, we performed linkage analysis and detected four linkage regions on chromosomes 1q23.2-24.1, 14q11.2-12, 15q26.2-26.3 and 17q24.3. Through whole exome sequencing, we found a de novo p.K1617del causative mutation in the <i>MYH7</i> gene. Therefore, we have diagnosed the disease as LDM, which is the first case in China. Our patients had more severe clinical manifestations by mimicking LGMD in comparison with the patients with the same mutation reported elsewhere.

Facioscapulohumeral Muscular Dystrophy: More Complex than it Appears.

Facioscapulohumeral muscular dystrophy (FSHD) has been classified as an autosomal dominant myopathy, linked to rearrangements in an array of 3.3 kb tandemly repeated DNA elements (D4Z4) located at the 4q subtelomere (4q35). For the last 20 years, the diagnosis of FSHD has been confirmed in clinical practice by the detection of one D4Z4 allele with a reduced number (≤8) of repeats at 4q35. Although wide inter- and intra-familial clinical variability was found in subjects carrying D4Z4 alleles of reduced size, this DNA testing has been considered highly sensitive and specific. However, several exceptions to this general rule have been reported. Specifically, FSHD families with asymptomatic relatives carrying D4Z4 reduced alleles, FSHD genealogies with subjects affected with other neuromuscular disorders and FSHD affected patients carrying D4Z4 alleles of normal size have been described. In order to explain these findings, it has been proposed that the reduction of D4Z4 repeats at 4q35 could be pathogenic only in certain chromosomal backgrounds, defined as “permissive” specific haplotypes. However, our most recent studies show that the current DNA signature of FSHD is a common polymorphism and that in FSHD families the risk of developing FSHD for carriers of D4Z4 reduced alleles (DRA) depends on additional factors besides the 4q35 locus. These findings highlight the necessity to re-evaluate the significance and the predictive value of DRA, not only for research but also in clinical practice. Further clinical and genetic analysis of FSHD families will be extremely important for studies aiming at dissecting the complexity of FSHD.

ZASP interacts with the mechanosensing protein Ankrd2 and p53 in the signalling network of striated muscle.

ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein.

Gene Expression Profiling in Tibial Muscular Dystrophy Reveals Unfolded Protein Response and Altered Autophagy

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.

DNAJB6 myopathy: A vacuolar myopathy with childhood onset

DNAJB6 mutations cause an autosomal dominant myopathy that can manifest as limb-girdle muscular dystrophy (LGMD1D/1E) or distal-predominant myopathy. In the majority of patients this myopathy manifests in adulthood and shows vacuolar changes on muscle biopsy. Clinical, electrophysiological, pathological, and molecular findings are reported. We report a 56-year-old woman, who, like 3 other family members, became symptomatic in childhood with slowly progressive limb-girdle muscle weakness, normal serum creatine kinase (CK) values, and myopathic electromyographic findings. Muscle biopsy showed vacuolar changes and congophilic inclusions, and molecular analysis revealed a pathogenic mutation in the DNAJB6 gene. Differences and similarities with previously described cases are assessed. Childhood-onset of DNAJB6 myopathy is more frequent than previously believed; congophilic inclusions may be present in the muscle of these patients.

CLINICAL ASPECTS AND MANAGEMENT OF MYOTONIC DYSTROPHIES

Myotonic dystrophy (DM) is the most common adult muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia and multiorgan involvement. To date two distinct forms caused by similar mutations have been identified. Myotonic dystrophy type 1 (DM1, Steinert’s disease) was described more than 100 years ago and is caused by a (CTG)n expansion in DMPK, while myotonic dystrophy type 2 (DM2) was identified only 18 years ago and is caused by a (CCTG)n expansion in ZNF9/CNBP. When transcribed into CUG/CCUG-containing RNA, mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, resulting in spliceopathy of downstream effector genes. Despite clinical and genetic similarities, DM1 and DM2 are distinct disorders requiring different diagnostic and management strategies. DM1 may present four different forms: congenital, early childhood, adult onset and late-onset oligosymptomatic DM1. Congenital DM1 is the most severe form of DM characterized by extreme muscle weakness and mental retardation. In DM2 the clinical phenotype is extremely variable and there are no distinct clinical subgroups. Congenital and childhood-onset forms are not present in DM2 and, in contrast to DM1, myotonia may be absent even on EMG. Due to the lack of awareness of the disease among clinicians, DM2 remains largely underdiagnosed. The delay in receiving the correct diagnosis after onset of first symptoms is very long in DM: on average more than 5 years for DM1 and more than 14 years for DM2 patients. The long delay in the diagnosis of DM causes unnecessary problems for the patients to manage their lives and anguish with uncertainty of prognosis and treatment.

A new disease allele for the p.C30071R mutation in titin causing hereditary myopathy with early respiratory failure

Hereditary myopathy with early respiratory failure is an autosomal dominant myopathy caused by mutations in the 119th fibronectin-3 domain of titin. To date all reported patients with the most common mutation in this domain (p.C30071R) appear to share ancestral disease alleles. We undertook this study of two families with the p.C30071R mutation to determine whether they share the same haplotype as previously reported British families or whether the mutation arose as a de novo event. We sequenced the 119th fibronectin-3 domain in these two probands and flanking polymorphisms associated with the British haplotype in hereditary myopathy with early respiratory failure. A family of Indian descent had a haplotype that was not compatible with the British shared haplotype. Cloning of the 119th fibronectin-3 domain in this patient demonstrated polymorphisms rs191484894 and novel noncoding variant c.90225C>T on the same allele as the mutation, which is distinct from previously reported British families. This proves that the p.C30071R mutation itself (rather than the haplotype containing this mutation) causes hereditary myopathy with early respiratory failure and suggests its independent origin in different ethnic groups.

P.12.2 Dominant distal myopathy due to slow channelopathy

Slow channel syndrome, first recognized by Engel in 1982, has distinct phenotypic features, dominant inheritance, selective weakness of cervical scapular and finger extensor weakness, ophtalmoparesis. Histochemical findings show type 1 fibers preponderance, small group of atrophic fibers of either fiber type, increased fiber size variability, tubular aggregates. We examined 3 biopsies in 3 patients (2 males – 55 years and 66 years, 1 female – 61 years) presenting a dominant distal myopathy with variable age at onset (from second to third decade). The patients had ophtalmoparesis, upper limb extensor, forearm and finger weakness and distal myopathy with a progressive slow course. The woman had also some difficulty in anterior tibial flexor muscle but also atrophy of forearms muscles. Electrophysiology on single stimulus of ulnar nerve showed repetitive CMAP. The genomic wide linkage analysis identified 3 regions co-segregating with the disease in chromosome 1, 13, 17. Next generation sequencing showed in all patients a known mutation in epsilon-subunit of AchR receptor and a mutation in phospholipase. Muscle showed mild changes consisting in atrophic fibers, mostly of type 2, some fiber type grouping, central nuclei and ring fibers. Such fibers are consistent with abnormal regeneration.We suggest that the occurrence of mutations involving both AchR receptor and phospholipase might concur in determining a new distal phenotype with permanent weakness. Our family was previously diagnosed as myotonic dystrophy or distal myopathy. The presence of the histochemical findings of both myopathy and neuropathy were present. We suggest the possibility of calcium overload due to prolonged open time of the AChR channel with a presynaptic component might determine a distal neuro-myopathy with slow evolution and muscle rearrangement due to a muscle fiber degeneration and functional denervation. The slow channel mutation should be searched in families with permanent distal weakness.

P9.17 Function of CCDC78 in muscle development and muscle disease

Congenital myopathies are a heterogeneous group of diseases that typically present in childhood with hypotonia and weakness and are commonly defined by characteristic changes observed on muscle biopsy. Currently, no treatments or disease-modifying therapies have been identified. Therapy development is hindered because approximately 40% of congenital myopathies are genetically unresolved. An overarching goal of our laboratory is to identify and characterize new causes of congenital myopathy. To this end, using whole exome sequencing we recently identified a splice-acceptor variant in the coiled-coil domain containing 78 gene (CCDC78) in a family with autosomal dominant core myopathy. CCDC78 is a previously uncharacterized gene with no known function that is highly enriched in skeletal muscle and localized to the perinuclear compartment and to the excitation contraction coupling apparatus. The specific goals of this study are to determine how mutation in CCDC78 results in muscle disease, establish the relationship between CCDC78 and other congenital myopathies, and to elucidate the normal function of CCDC78. To accomplish these goals we have created both morpholino based and stable genetic models of CCDC78 mutation in the zebrafish, including characterization of a newly developed loss of function CCDC78 mutant (sa3254). We have combined analysis of these zebrafish models with cell culture studies and examination of a range of human muscle biopsies. Current data suggests that CCDC78 participates in both the regulation of excitation-contraction coupling and in the regulation of autophagy. Additional ongoing experimentation will examine expression of CCDC78 in other congenital muscle diseases, will determine interacting partners for CCDC78, and will establish the normal function for CCDC78 in skeletal muscle development and homeostasis. In all, we present the first extensive characterization of this newly identified congenital myopathy gene.

O.22 Autosomal dominant Core Congenital Myopathy caused by a mutation in the MYH7 gene

Autosomal dominant (AD) Central core disease (CCD) named currently congenital myopathy with core is an inherited disorder characterised by the presence of cores which are well-limited rounded areas devoid of any oxidative staining. The cores extend almost along the full length of the fibres; these areas correspond to sarcomeric disorganisation, Z line streaming and absence of mitochondria. Genetic studies of AD-CCD families demonstrated the presence of heterozygous mutations in the RYR1 gene in the large majority of families. However, this gene was excluded in some families, suggesting a genetic heterogeneity of AD-Core myopathies (Romero et al., 2005). To enlarge the genetic spectrum of autosomal dominant congenital myopathies with cores demonstrating mutations in a second gene. AD-CCD family with three affected members: the mother and two of three siblings. The symptoms began during the early childhood with delayed motor development. Later they develop proximal weakness, hypertrophy of calves, scapular winging and significant weakness (amyotrophic) of quadriceps and tibialis anterior. No cardiac or ocular involvement was noted. The muscle biopsies sections showed a particular pattern: relatively large and eccentric cores (placed near the subsarcolemmal regions) associated with type 1 predominance and fibre type disproportion in a patient. The large majority of the cores have abrupt borders. Electron microscopy confirmed the presence of multiple large disorganised sarcomeric areas as characteristic unstructured and structured cores. Moreover, some fibres contain focal disorganised areas. Exome sequencing analysis identified a heterozygous missense mutation L1723P in MYH7 segregating with the disease and affecting a conserved residue in the tail domain. With this study we describe MYH7 as a second causative gene for AD-CCD in addition to RYR1 . Our study enlarges the genetic spectrum of AD-CCD myopathies.

P.9.4

Nemaline myopathy is one of the most common forms of congenital myopathy characterized pathologically by presence of nemaline rods in muscle fibers. Clinical features are quite variable from severe infantile form to adult onset milder form. Genetic diagnosis is not easy because several causative genes have been identified including a very much big gene like NEB . To know the frequency and clinical and pathological characteristics of each causative genes of nemaline myopathy. We chose more than 200 patients who were pathologically diagnosed to have nemaline myopathy from muscle repository in the National Center of Neurology and Psychiatry, Japan. Mutation screening is on going using a combination of the target re-sequencing by next generation sequencer and the direct sequencing by Sanger method. Mutations in ACTA1 were identified in 15.4% of the patients examined in our cohort. Clinically, most of the patients carrying ACTA1 mutation were sporadic and showed severe infantile form, but a family with autosomal dominant mild myopathy was also found. We identified many variants in NEB , RYR1 , and other causative genes in more than one third of our cohorts, but only one variant was found in many of these patients. Presence of unidentified genes was suggested because no mutation in the known causative genes was found in a half of nemaline myopathy. Other disorders can also produce pathological findings consistent with nemaline myopathy.

P.15.2 Elucidating the mechanism of disease in BAG3 related myofibrillar myopathy

Myofibrillar myopathies are a group of chronic muscle diseases characterised at the cellular level by failure of the muscle fibre at the Z-disk and accumulation of protein aggregates. The vast majority of these diseases are late onset dominant disorders with symptoms typically evident between 35 and 50 years of age. Patients suffer progressive muscle weakness over many years and have reduced life expectancy due to respiratory muscle failure and cardiac complications. Whilst structural failure of the muscle fibre is a feature of myofibrillar myopathies not all of the proteins associated with myofibrillar myopathy have a structural role. Recently mutations in BAG3, a co-chaperone with no direct role in muscle function, were identified as a cause of this disorder. This discovery opened a new area of investigation into the role of chaperones in myopathies and the mechanism of disease in myofibrillar myopathy. In order to investigate the role of BAG3 in muscle development and disease we examined zebrafish lacking BAG3 and those with ectopic expression of GFP-tagged forms of either wildtype BAG3 or the dominant myofibrillar myopathy mutant form Bag3P209L. Examination of these fish identified both the fibre failure and the formation of protein aggregates characteristic of myofibrillar myopathy. Utilising the advantage of the zebrafish model system to examine the onset and progression of the phenotype in vivo, combined with detailed characterisation by immunolabelling and confocal microscopy, we were able to develop a novel model that explains the mechanism of disease in BAG3 related myofibrillar myopathy. We will present this model and discuss its implications for other forms of myofibrillar myopathy.

P.5.19 Fhl1 W122S knock-in mice manifest late-onset mild myopathy

FHL1 is a member of the four-and-a half-LIM (FHL) domain protein family and it is highly expressed in adult human skeletal muscle. It is believed to participate in sarcomere assembly, muscle growth and differentiation, and in the biomechanical stress responses. Mutations in FHL1 gene have been associated with different myopathies, including reducing body myopathy, scapuloperoneal (SP) myopathy, X-linked myopathy with postural muscle atrophy, rigid spine syndrome (RSS), and Emery-Dreifuss muscular dystrophy. In 2008, we identified a missense mutation in the second LIM domain of FHL1 (c.365 G>C, p.W122S) in a family with SP myopathy. We have generated a knock-in mouse model harboring the c.365 G>C Fhl1 mutation and have investigated the effects of this mutation at pre-symptomatic, phenotypic-onset, and late-stage of the disease, in hemizygous males and heterozygous females mice. Survival was comparable in mutant and wild-type animals. Adult hemizygous males mice showed a slowly progressive phenotype similar to human patients with relatively late-onset muscle weakness. In particular, we observed reduced forelimb strength and exercise capacity. Western blot analysis showed absence of FHL1 protein in muscle of hemizygous males at advanced stages. This animal model may help to elucidate the role of FHL1 in skeletal muscle and the pathomechanism of FHL1 mutations in X-linked dominant scapuloperoneal myopathy.