Pedigree of SPM Family C
Dark symbols indicate affected individuals. Genotypes are listed below each tested individual (two clinically unaffected are not shown as they requested). Haplotypes shared among the affected individuals are boxed. Individuals are numbered according to a prior publication. 6

FHL1 Protein Expression in SPM Patients
Western blot analysis of FHL1 and vinculin in skeletal muscle of four patients (P1, III-24; P2, III-14; P3, III-32; and P4, IV-18) with the W122S FHL1 mutation and two controls.

Evolutionary Conservation of Tryptophan-122 in FHL1
Tryptophan at amino acid 122 (arrow) in human FHL1 is highly conserved across species.

Structural Analysis of the FHL1 W122S Mutation
(A) Sequence alignment of the 4 LIM domains in FHL1 demonstrates 13 conserved amino acids (highlighted in red) including 8 residues (indicated by asterisks) that coordinate the two zinc ions. The secondary structure indicated on the first line is derived from an NMR model containing the second LIM domain (PDB, 1X63). Amino acid numbers refer to the second LIM domain. An arrow indicates the position of the disease-associated W122S mutation.
(B) The structure of LIM domain 2 of FHL1 is shown as a cartoon diagram. Wild-type residue W122 (in cyan) is replaced by serine (in magenta) in affected individuals. A molecular surface calculated for the mutant protein with GRASP 26 and colored to demonstrate the predicted electrostatic potential (blue is positive charge and red is negative). Substitution of the normal Trp residue by a smaller Ser side chain is likely to change the surface of the protein. Side chains of the residues that coordinate the zinc ions (shown as spheres) are shown in stick representation, as well as the nearby conserved F127.
(C) Apparent van der Waals contacts between wild-type W122 with neighboring F127, T120, and K107 are shown as overlapping clouds of small spheres in an image generated with PYMOL (DeLano Scientific LLC).