Exome sequencing reveals a nebulin nonsense mutation in a dog model of nemaline myopathy.

Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.

Nemaline myopathy in a six-month-old Pomeranian dog.

ABSTRACT: Nemaline myopathy - a clinically and genetically complex heterogenous group of disorders - is described uncommonly in humans and rarely in animals, and is characterised by progressive muscle weakness. The diagnosis is confirmed by histological and/or ultrastructural identification of subsarcolemmal, thread-like, rod-shaped structures called nemaline rod bodies within more than 40% of skeletal muscle fibres. These rods contain the Z-line protein, α-actinin, that can be effectively stained in skeletal muscles using Gomori or Masson trichrome and negatively stained with periodic acid-Schiff. Similar rod-like bodies have been found in smaller numbers in dogs with endocrine disorders and occasionally in other conditions in humans. This report is of a six-monthold Pomeranian dog which had progressive exercise intolerance over a two-month period associated with severe disuse muscle atrophy of the thoracic limbs, as well as gradual pelvic limb weakness and regurgitation of food. Baseline diagnostics ruled out endocrinopathies and after histological and ultrastructural evaluation of thoracic limb muscles and nerve biopsies confirmed nemaline myopathy. The clinical course, diagnostic test results, ultrastructure of skeletal muscle and peripheral nerve, gross necropsy findings and histopathology using various stains are described and illustrated.

Sarcomeric-alpha-actinin defective in vinculin-binding causes Z-line expansion and nemaline-like body formation in cultured chick myotubes.

The Z-line in each striated muscle has a precisely defined width that corresponds to muscle fiber type, and it can enlarge several fold in nemaline myopathy. To explore the mechanism(s) underlying Z-line width and structure maintenance, a series of sarcomeric-alpha-actinin mutants tagged with myc-epitope was transfected into cultured chick myotubes. By double-staining transfected myotubes with myc and myofibrillar protein antibodies, we found that alpha-actinin mutants with deletion of the region from the beginning of the fourth spectrin repeat to the start of the EF-hands resulted in expansion of Z-line width, often displayed a doublet staining pattern, and resulted in formation of nemaline-like bodies in older myotubes under fluorescence microscope. Yeast-two hybridization analysis demonstrated that this region was involved in vinculin binding, and for vinculin to bind alpha-actinin, residues 1-116 and 258-323 were required. Hence, we have defined a critical region of s-alpha-actinin that affects the width and integrity of the Z-line. This region is at least involved in the interaction with vinculin.

Centronuclear Myopathy with Abundant Nemaline Rods in a Japanese Black and Hereford Crossbred Calf.

Histopathological examination was performed on skeletal and diaphragmatic muscles from an 8-month-old male crossbred calf showing abnormal gait and tremor of the hindlimbs. There were numerous round fibres with centrally placed nuclei forming nuclear chains in longitudinal sections, associated with interstitial fibrosis or adipose tissue infiltration. On nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) staining, some muscle fibres in severe lesions showed a spoke-like appearance due to a radial arrangement of sarcoplasmic strands. Additionally, increased NADH-TR activity in the subsarcolemmal structures, appearingas ring-like or necklace-like forms, were observed. Transmission electron microscopy revealed dilated sarcoplasmic reticulum and variably shaped electron-dense inclusions consisting of myofibrillar streams. Another prominent feature was the existence of numerous nemaline rods within muscle fibres; these were stained red by Gomori's trichrome stain. Immunohistochemistry revealed that the nemaline rods showed strong immunoreactivity with α-actinin and desmin antibodies. Electron microscopically, these structures were composed of dense-homogeneous material and continuous with the Z disk. The case was diagnosed as centronuclear myopathy with increased nemaline rods.

Congenital myopathy with abundant nemaline rods in a cat.

Nemaline myopathy is associated with rod-shaped structures in muscle fibers. At least seven distinct clinical forms have been described in humans and mutations have been identified in five different thin-filament genes. Only a few cases of spontaneously occurring nemaline myopathy have been reported in animals and include an adult-onset form in a family of cats and an early-onset form in a dog. Here, we describe a 2-year-old male, neutered, domestic shorthaired cat that was referred to the Veterinary Medical Teaching Hospital, University of California-Davis, for evaluation of chronic, progressive weakness, and fine tremors. Neurologic deficits were restricted to the neuromuscular system. Electromyography showed mild to moderate diffuse spontaneous activity. Although rod bodies were prominent on light and electron microscopic evaluation of biopsies from several muscles, sarcoplasmic accumulations of dystrophin, desmin, and spectrin also were identified by immunohistochemistry. These findings may represent the occurrence of rod bodies in conjunction with a protein-aggregate myopathy.

Nemaline rods in canine myopathies: 4 case reports and literature review.

The diagnosis of nemaline rod myopathy (NM) is based on the presence of numerous pathognomonic rods within a fresh frozen muscle biopsy specimen. Three forms of congenital NM have been described in humans, and rods have been found to occur in various other conditions. A similar myopathy was described in 1986 in a family of cats. In this report, we describe a case of congenital NM in a 10-month-old Border Collie, an adult-onset NM in an 11-year-old Schipperke, and 2 acquired myopathies with nemaline rods in adult dogs associated with hypothyroidism and Cushing's syndrome. Common clinical features included exercise intolerance, abnormal electromyography, and the presence of nemaline rods in fresh, frozen, and glutaraldehyde-fixed biopsies from proximal appendicular limb muscles. Staining of cryostat sections of muscle biopsy specimens by the modified Gomori trichrome technique disclosed numerous rod bodies that were localized to type 1 fibers by the histochemical adenosine triphosphatase reaction. Accumulation of rods also was demonstrated by electron microscopy in 2 of the cases with localized enlargement and streaming of Z lines. Documentation of NM in a young Border Collie and the adult-onset form in the Schipperke alerts clinicians to the existence of this disorder in these breeds.

Adult-onset nemaline myopathy in a dog presenting with persistent atrial standstill and primary hypothyroidism.

A nine-year-old neutered female mixed breed dog presented for evaluation following a five-day history of lethargy, inappetence, weakness, abdominal distension and generalised muscle atrophy. Persistent vatrial standstill with a junctional rhythm was identified on electrocardiogram. Echocardiogram identified moderate dilation of all cardiac chambers and mild thickening of the mitral and tricuspid valves. Serology was negative for Neospora caninum and Toxoplasma gondii. Permanent pacemaker implantation was performed in addition to endomyocardial and skeletal muscle biopsies. Cryosections from the biceps femoris muscle showed numerous nemaline rod bodies while endomyocardial biopsies were possibly consistent with end-stage myocarditis. Rod bodies have rarely been reported in the veterinary literature. To the authors' knowledge, this is the first report of adult-onset nemaline rod myopathy and hypothyroidism with concurrent cardiac disease in a dog.