Characterization of the R7S mutation of Heat Shock Protein HSPB3 and of two novel mutations found in patients suffering of myopathy: understanding the mechanisms leading to disease.

HSPB3 is a poorly characterized member of the small HSP/HSPB family (HSPB1-HSPB10) that forms a complex with HSPB2 with a defined 1:3 ratio. The HSPB2/HSPB3 complex is induced during muscle differentiation and plays a role in muscle maintenance. Recently the R7S mutation in HSPB3 has been associated with distal hereditary motor neuropathy type 2C (dHMN 2C). Here we report the identification in myopathic patients of two novel mutations in HSPB3: 1) one mutation affects the R116 residue, which corresponds to a key amino acid in the alpha-crystallin domain, whose mutation in other members of the HSPB family also causes disease (it is equivalent to e.g. R120 in HSPB5, whose mutation into G causes MFM and to K141 in HSPB8, whose mutation into E or N causes dHMN); 2) the other mutation disrupts the reading frame leading to a premature stop codon at amino acid 50. Both mutations were not found in more than 400 normal alleles. Expression studies allowed us to confirm that the mutation causing a premature stop codon leads to the generation of an unstable protein that is likely immediately degraded after synthesis and cannot be detected. Also, while both expressed, the R7S mutant was more stable than the R116 one. We next characterized in cells and in vitro the ability of these HSPB3 mutants to interact with HSPB2 and form the HSPB2/HSPB3 complex. We found that while the R7S mutant of HSPB3 was still able to interact with HSPB2, the R116 mutant was not. Future studies will allow us to better characterize how these HSPB3 mutants affect HSPB3 and, indirectly, HSPB2 stability, subcellular localization and function. They will also elucidate on HSPB3 and HSPB2 function in both motor neurons and myoblasts and will shed light on how mechanistically the mutations in HSPB3 affect the function and viability of these cell types, contributing to disease.