NonHumanModel: Mouse (Mus musculus)
GeneAlteration: CRISPR-Cas9 gene editing was used to produce gene-edited mouse zygotes. This was accomplished by designing a guide RNA (gRNA) with the CRISPR design tool and microinjecting the gRNA complex along with Cas9 mRNA and a ultramer repair template including the single deletion into mouse zygotes. This directs double-strand breaks in the genome with homology directed repair causing the precise genome edit necessary. These were injected into psuedo-pregnant C57BL/6J mice. After birth, tail DNA was genotyped via PCR and Sanger seuqencing to confirm the mutation. One heterozygous 525delT mouse was crossed and the offspring backcrossed onto DBA/2J background until the F5 generation confirmed homozygous for the severe LTBP4 allele which results in a more severe disease progression. RT-PCR confirmed the presence of the deletion and loss of γ-sarcoglycan was confirmed via immunoblot analysis and immunofluorescence microscopy.
ModelPhenotype: HP:0003202, HP:0031237, HP:0030951, HP:0003713, HP:0011987, HP:0003557, HP:0003236, HP:0003323, HP:0005957 (Skeletal muscle atrophy, Internally nucleated skeletal muscle fibers, Skeletal muscle fibrosis, Muscle fiber necrosis, Ectopic ossification in muscle tissue, Increased variability in muscle fiber diameter, Progressive muscle weakness, Breathing dysregulation)
ModelFreeText: Decreased cross-sectional area of myofibers, Increased immune infiltrate, Increased dye uptake into compromised myofibers
HumanPhenotype: HP:0003202, HP:0030951, HP:0003713, HP:0003236, HP:0003323, HP:0005957 (Skeletal muscle atrophy, Skeletal muscle fibrosis, Muscle fiber necrosis, Elevated serum creatine kinase, Progressive muscle weakness, Breathing dysregulation)
HumanFreeText: N/A
ModelEvidence: This model has many positive aspects compared to the default mouse model, although it also has some challenges to overcome. In terms of the generation, CRISPR-Cas9 gene editing via HDR is a relatively simple way to modify the genome as needed and create only the variant of interest while leaving the rest untouched. This results in a knock-in with a reasonable assurance that whatever phenotypes are displayed stem solely from the change in the SGCG gene. Specifically the muscle atrophy, elevated serum creatine kinase, progressive weakness (half the strength of controls at 4 months), and signifiacntly increased muscle fibrosis/necrosis are all hallmarks of the human LGMD presentation and are recapitulated here. However, the presence of a known muscular dystrophy-modifying background raises the question of whether some of these phenotypes would not be seen in a mouse with the C57BL/6J background. Unfortunately this is not answered in the paper, so it introduces some doubt as to the total validity of the results.
ModelVariant: NM_000231.2(SGCG):c.525del (p.Phe175fs)
ClinVar: 189243 https://www.ncbi.nlm.nih.gov/clinvar/variation/189243/