Proteomic identification of the LIM domain protein FHL1 as the gene-product mutated in reducing body myopathy

Reducing body myopathy (RBM) is a rare progressive myopathy defined by characteristic aggresome-like inclusions in muscle. Traditional gene identification in this disease has been complicated by the frequently sporadic occurrence of patients and small family sizes. As an alternative approach we used laser microdissection of the intracytoplasmic inclusions followed by nanoflow LC-MS/MS coupled with proteomic analysis, to identify FHL1 (SLIM1), encoded on Xq26.3, as the most prominent component of the inclusions. FHL1 belongs to the family of four and a half LIM domain proteins (FHL1–5); it is predominantly expressed in skeletal but also in cardiac muscle. Mutational analysis up to now identified 7 different FHL1 mutations in 11 patients with RBM, including severely affected sporadic female and male patients as well as 2 familial cases with severely affected boys and more mildly affected mothers. Mutations were found exclusively in the second LIM domain, changing invariant in the zinc-coordinating histidine and cysteine residues. Transfection of mutant FHL1 induced the formation of inclusions with aggresomal features in COS-7 and skeletal muscle C2C12 cells, incorporating both mutant and wild type FHL1 as well as trapping other proteins in a dominant negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de-novo mutations in FHL1 thereby defining a new X-linked protein aggregation disorder of muscle.