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

Abstract

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.

Fig. 1

Histopathology establishes NM. Cryosections from the triceps muscle of an affected pup (a–c) and archived control triceps muscle (d–f) were stained with H&E (a, d), modified Gomori trichrome (b, e) and following incubation with monoclonal antibodies against type 1 and type 2 myosin heavy chains (c, f). Excessive variability in myofiber size and atrophy were observed in the affected muscle with the H&E stain (a) compared to control muscle (d). Numerous myofibers in the affected muscle contained rod bodies (b, arrows) not evident in control muscle (e). Both type 1 and type 2 fibers were atrophic (c) with fibers of both fiber types similar in size in control muscle (f). Bar 50 µm for images a–f. By electron microscopy, numerous electron dense rods were apparent along the long axis parallel to that of the muscle fiber (g). The rods were in structural continuity with Z disks (h), had the same electron density as the Z lines of adjacent sarcomeres, and had a similar lattice pattern of periodic cross striations. Bar 0.18 µm for images g and h

Fig. 2

Filtering of NEB variants reveals S8042X. a Filtering parameters are shown to the left, with the number of rejected variants to the right. Total variants prior to each filtering parameter are shown above the arrows, beginning with the total number of variants. b Chromatograms from Sanger sequencing show the wild-type and mutant alleles

Fig. 3

Nebulin is drastically reduced in skeletal muscles with S8042X. a Protein analysis on a 2–7 % gradient acrylamide gel reveals a clear loss of nebulin in muscles from affected dogs compared to healthy controls. b Western blots for the same samples using antibodies to nebulin’s N- and C-terminus. c and d Average quantification (CTRL 1 and CTRL 2; AFF 1 and AFF 2) of western blots, indicating a severe loss of nebulin protein in affected dogs.