A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation-prone Ano5 peptide.

Mutations in ANO5 cause several human diseases including gnathodiaphyseal dysplasia 1 (GDD1), limb-girdle muscular dystrophy 2L (LGMD2L), and Miyoshi myopathy 3 (MMD3). Previous work showed that complete genetic disruption of Ano5 in mice did not recapitulate human muscular dystrophy, while residual expression of mutant Ano5 in a gene trapped mouse developed muscular dystrophy with defective membrane repair. This suggests that truncated Ano5 expression may be pathogenic. Here, we screened a panel of commercial anti-Ano5 antibodies using a recombinant adenovirus expressing human Ano5 with FLAG and YFP at the N- and C-terminus, respectively. The monoclonal antibody (mAb) N421A/85 was found to specifically detect human Ano5 by immunoblotting and immunofluorescence staining. The antigen epitope was mapped to a region of 28 residues within the N-terminus. Immunofluorescence staining of muscle cryosections from healthy control subjects showed that Ano5 is localized at the sarcoplasmic reticulum. The muscle biopsy from a LGMD2L patient homozygous for the c.191dupA mutation showed no Ano5 signal, confirming the specificity of the N421A/85 antibody. Surprisingly, strong Ano5 signal was detected in a patient with compound heterozygous mutations (c.191dupA and a novel splice donor site variant c.363 + 4A > G at the exon 6-intron 6 junction). Interestingly, insertion of the mutant intron 6, but not the wild-type intron 6, into human ANO5 cDNA resulted in a major transcript that carried the first 158-bp of intron 6. Transfection of the construct encoding the first 121 amino acids into C2C12 cells resulted in protein aggregate formation, suggesting that aggregate-forming Ano5 peptide may contribute to the pathogenesis of muscular dystrophy.