Congenital muscular dystrophy in a dog with a LAMA2 gene deletion

Abstract A 2‐year‐old female spayed dog was presented with a chronic history of short‐strided gait and inability to completely open the jaw. Clinical signs were present since the dog was adopted from a humane society at a few months of age. Serum creatine kinase activity was abnormally high. Neurological examination, electromyography, muscle biopsies with immunofluorescent staining, and whole genome sequencing (WGS) were performed. A dystrophic phenotype was identified histologically in muscle biopsies, deficiency of laminin α2 protein was confirmed by immunofluorescent staining, and a deletion in the LAMA2 gene was identified by analysis of the WGS data. Congenital muscular dystrophy associated with a disease variant in LAMA2 was identified.


| INTRODUCTION
Congenital muscular dystrophies (CMDs; muscle disease characteristic of a dystrophic process but not including dystrophies associated with mutations in dystrophin or dystrophin associated proteins) and congenital myopathies (nondystrophic myopathies with characteristic histological and histochemical findings) are the most common groups of congenital onset muscle diseases in humans 1 and are similarly characterized and classified in veterinary medicine. 2 Examples of CMDs in humans include but are not limited to myopathies associated with mutations in COL6 (COL6A1, A2, and A3), LAMA2 (laminin α2 chain), (LARGE and other disorders of hypoglycosylation (α-dystroglycanopathies), SEPN1 and RYR1 related myopathies. In veterinary medicine, specific mutations have been identified in COL6A3 in Labrador retrievers, 3 COL6A1 in Landseer dogs, 4 and recently in Labrador retrievers with α-dystroglycanopathy and a mutation within LARGE1. 5 Laminins are large glycoproteins that are structural components of basement membranes in many tissues. In muscle, laminins form the specialized extracellular matrix that immediately abuts and surrounds each muscle fiber. 6 Laminins are heterotrimeric proteins composed of α, β, and γ subunits that are connected to the sarcolemma via the dystrophin-glycoprotein complex 6,7 and integrins. 8 Multiple isoforms of each type of subunit exist that enable formation of many, sometimes tissue-specific laminins. The primary heterotrimers present in skeletal muscle are laminin 2 (α2-β1-γ1) and laminin 4 (α2-β2-γ1) also collectively referred to as merosin. Laminin α2 is also found in the basement membranes of Schwann cells where it is thought to play a role in the ensheathment and myelination of peripheral nerves, 9 in brain blood vessels, 10 and in other tissues.
The LAMA2 gene encodes the α2 subunit of the heterotrimeric laminin-2 complex, in which predominantly recessive LAMA2 variants are the basis for a CMD subtype termed the laminin α2-related muscular dystrophies. Deficiency in laminin α2 protein determined with immunofluorescent staining of muscle cryosections has been reported in humans, 11 a mouse model, 12 3 cats, 13,14 and in a dog. 15 Here we describe a disease variant in LAMA2 in an additional dog resulting in laminin α2-deficient CMD. Staining for laminin α2 localization was not detectable in cryosections from the affected dog but normal in control archived muscle ( Figure 1C). Staining for other extracellular matrix proteins including laminin γ1 subunit and collagen VI was similar to controls ( Figure 1C).

| CASE REPORT: WHOLE GENOME SEQUENCING AND DATA ANALYSIS
A polymerase chain reaction-free library was prepared using DNA extracted from muscle of the affected dog and sequenced in 1 lane of an Illumina HiSeq 4000 sequencer by GeneWiz (South Plainfield, New Jersey). The reads were mapped against the dog reference genome assembly (CanFam3.1) as described 18,19 Figure 2). This deletion was observed as an absence of read alignments over this region in the case sequence data, while abundant reads were aligned from the control sequence data.

| DISCUSSION
The muscularis dystrophia mouse (dy/dy), reported in 1955, 20 was the first animal model of muscular dystrophy. Deficiency of laminin α2 and a specific mutation was identified in dy2J/dy2J mice 40 years later. 12 Since that time, laminin α2 protein deficiency has been identified in a dog 15 and in cats 13,14 but until now a specific mutation in LAMA2 for cats or dogs has not been reported. Here we describe a deletion encompassing exon 5 of the LAMA2 gene that is the apparent cause of a laminin α2-deficient CMD in a dog.
The laminin α2-related muscular dystrophies in humans are typically autosomal recessive. The dog we report was obtained from a shelter and neither a pedigree nor information on family members was available. However, we speculate that this was also an autosomal Therefore, future studies of LAMA2 CMDs in dogs and cats should include these electrodiagnostic and tissue evaluations.
In human patients with LAMA2 related CMD, subclinical cardiac involvement is described, notably right bundle branch block and left ventricular dysfunction, with rare reports of heart failure. 11 Regular cardiac monitoring with cardiac rhythm assessment by Holter monitoring and cardiac imaging by echocardiogram has been recommended for all LAMA2 patients. A pronounced sinus arrhythmia was noted in this dog but with no evidence of cardiomyopathy. Cardiomyopathy is F I G U R E 2 LAMA2 deletion detected with whole genome sequencing. A 3957 bp segment flanking the 67 735 500 bp position on canine chromosome 1 is shown. Whole genome sequence reads obtained from a normal dog (upper set) and the case (lower set) were aligned to the CanFam3.1 reference genome. Each sequence read that aligns to this region is depicted as a short gray bar (the length of each gray bar is approximately 100 bp) and the sum of reads over any given bp is indicated by the "gray wave" above each dog's set of mapped reads. The absence of any reads aligning to the approximate 67 734 500 to 67 736 500 interval signifies the deletion of that segment in the case dog's genomic DNA. The position of the 180 bp exon 5 in the reference sequence is indicated by the blue box on the bottom relatively common in dystrophin-deficient muscular dystrophy 23 but information is limited in the LAMA2 CMD in dogs.