FLNC-Associated Myofibrillar Myopathy

Figure 1. Pedigree and p.K2676Pfs*3 FLNC Mutation Analysis (A) Pedigree of the German family with LGMD-like MFM identified in this study associated with a mutation of FLNC exon 48. The index patient III:8 is indicated by an arrow. Individuals with proven mutation and deceased family members who had muscle weakness are represented by filled symbols. (B) Mutation detection via Sanger sequencing with subsequent translation to amino acids in silico.

Figure 2. Effect of p.K2676Pfs*3 FLNC Mutation (A and B) Sketch illustrating the effect of the mutation. Topology of filamin C domain 24 as derived from its crystal structure17 aligned to the amino acid sequence (A) and in a 2-dimensional view (B). β-strands (C and D) form the interface between dimerized domains. Strand G directly interacts with strand (A), which is important for the coherence of the 2 β-sheets that comprise an Ig-like domain. The position of the p.K2676Pfs*3 mutation is marked with a red asterisk. It results in the loss of β-strands (D–G) rendering a correct folding of the domain impossible. The gray asterisk marks the position of the p.W2710X mutation. (C) RT-PCR of control and patient cDNA digested with AleI. cDNA reverse transcribed from control and patient skeletal muscle mRNA amplified with 2 different primer pairs and digested with AleI. Because control cDNA does not contain an AleI restriction site, only mutant cDNA was digested into 160 and 170 bp fragments (primer pair 1) and 450 and 138 bp fragments (primer pair 2), respectively. In patient skeletal muscle, wild-type and mutant mRNA are expressed in an approximate ratio of 4:3.

Figure 3. Muscle Imaging Findings in Lower Extremities T1-weighted muscle MRI revealed severe fatty degenerative changes in patients III:4 and III:5 with a disease duration of more than 10 years. The gracilis and the lateral head of the gastrocnemius were relatively spared in both patients, as indicated by arrowheads. Mild early changes (arrows) were seen either in the tibialis anterior and semimembranosus (patient III:6) or in the soleus muscle (patients III:7 and III:8).

Figure 4. Histochemical Findings and Immunolocalization Studies Analysis of muscle biopsy samples from patients III:4 (A and C) and III:7 (B, D, and E). (A.a and B.c) Cryosections stained with H&E, TC, and NADH showed fiber diameter variability, fiber splitting, adipose replacement, endomysial fibrosis, increase in central nuclei, and marked irregularities of the intermyofibrillar network with many fibers showing multiple areas devoid of oxidative enzyme activity. These findings were more pronounced in III:4. (C.a–D.c) Cryosections double stained with antibodies recognizing desmin (DES) and FLNc to localize protein aggregations. Bar: 50 μm. (E.a–E.c) Longitudinal cryosections were double stained for DES and FLNc. Both proteins colocalized in aggregates (arrows), but also in microlesions (arrowheads). Bar: 10 μm.

Figure 5. Proteomic Analysis of Skeletal Muscle Samples From Patients With MFM-Filaminopathy Aggregate and intraindividual control samples were collected by laser microdissection. The bar chart in (A) shows the proportion of proteins identified as overrepresented in aggregate samples (compared with controls), grouped by function. Z-disc and Z-disc–associated proteins were the most abundant components followed by proteins involved in protein quality control and degradation. (B) Proteomic profiles of overrepresented aggregate proteins in patients with filaminopathy caused by different FLNC mutations. Pie charts illustrate that the pattern in patients with p.K2676Pfs*3 mutation was highly similar to that in patient with p.W2710X or p.V930_T933del mutation. FLNc was always the most abundant protein, followed by desmin and the FLNc binding partners Xin actin-binding repeat-containing protein 2 (XIRP2) and nebulin-related-anchoring protein (N-RAP). Proportions of αB crystallin, Xin actin-binding repeat-containing protein 1 (Xin), obscurin, and nestin were also similar.

Figure 6. Functional Studies of the p.K2676Pfs*3 Mutation (A) Ribbon representation of the human FLNc domain 24 structure (PDB code 1V05). The d24 dimer is shown in blue with regions missing in the p.K2676Pfs*3 mutant variant shown in light orange. β-strands as well as N and C termini are indicated. Asterisks distinguish β-strands of individual subunits. The mutation leads to loss of β-strands involved in the formation of 2 extended antiparallel β-sheets (B-E-D-D*-E*-B* and C-F-G-G*F*-C*), which stabilize domain fold integrity as well as the dimer, leading to severely impaired dimerization. (B) Circular dichroism spectroscopy performed with purified, bacterially expressed wild-type and p.K2676Pfs*3 mutant FLNc d23–24 constructs. The mean residue ellipticity (ΔεMR) of wild-type and mutant FLNc shows a minimum at 218 nm characteristic of a β-structure. However, the deletion mutant displays a lower amplitude at this wavelength, indicating loss of β-structure and/or folding. (C) Thermolysin digestion of wild-type and mutant FLNc d23–24 for 1–20 minutes. The mutant protein was completely digested after 20 min, whereas almost all the wild-type variant was still intact, and the p.W2710X was only…