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Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications. [ABSTRACT FROM AUTHOR]

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations. [ABSTRACT FROM AUTHOR]

Mutations in the skeletal muscle α-actin gene (ACTA1) cause a range of pathologically defined congenital myopathies. Most patients have dominant mutations and experience severe skeletal muscle weakness, dying within one year of birth. To determine mutant ACTA1 pathobiology, transgenic mice expressing ACTA1(D286G) were created. These Tg(ACTA1)D286G mice were less active than wild-type individuals. Their skeletal muscles were significantly weaker by in vitro analyses and showed various pathological lesions reminiscent of human patients, however they had a normal lifespan. Mass spectrometry revealed skeletal muscles from Tg(ACTA1)D286G mice contained ∼25% ACTA1(D286G) protein. Tg(ACTA1)D286G mice were crossed with hemizygous Acta1+/− knock-out mice to generate Tg(ACTA1)D286G+/+.Acta1+/– offspring that were homozygous for the transgene and hemizygous for the endogenous skeletal muscle α-actin gene. Akin to most human patients, skeletal muscles from these offspring contained approximately equal proportions of ACTA1(D286G) and wild-type actin. Strikingly, the majority of these mice presented with severe immobility between postnatal Days 8 and 17, requiring euthanasia. Their skeletal muscles contained extensive structural abnormalities as identified in severely affected human patients, including nemaline bodies, actin accumulations and widespread sarcomeric disarray. Therefore we have created valuable mouse models, one of mild dominant ACTA1 disease [Tg(ACTA1)D286G], and the other of severe disease, with a dramatically shortened lifespan [Tg(ACTA1)D286G+/+.Acta1+/–]. The correlation between mutant ACTA1 protein load and disease severity parallels effects in ACTA1 families and suggests altering this ratio in patient muscle may be a therapy for patients with dominant ACTA1 disease. Furthermore, ringbinden fibres were observed in these mouse models. The presence of such features suggests that perhaps patients with ringbinden of unknown genetic origin should be considered for ACTA1 mutation screening. This is the first experimental, as opposed to observational, evidence that mutant protein load determines the severity of ACTA1 disease. [ABSTRACT FROM AUTHOR]