New family with HSPB 8-associated autosomal dominant rimmed vacuolar myopathy

Abstract

Objective: We clinically and molecularly characterize a new family with autosomal dominant rimmed vacuolar myopathy (RVM) caused by mutations in the HSPB8 gene.

Methods: We performed whole-exome and whole-genome sequencing in the family. Western blot and immunocytochemistry were used to analyze 3 patient fibroblasts, and findings were compared with their age- and sex-matched controls.

Results: Affected patients have distal and proximal myopathy, with muscle biopsy showing rimmed vacuoles, muscle fiber atrophy, and endomysial fibrosis typical of RVM. Muscle MRI showed severe relatively symmetric multifocal fatty degenerative changes of the lower extremities. We identified a duplication of C at position 515 of the HSPB8 gene (c.515dupC) by whole-genome sequencing, which caused a frameshift with a predicted alternate stop codon p.P173SFS*43 in all affected individuals, resulting in an elongated protein product. Western blot and immunocytochemistry studies revealed reduced expression of heat shock protein beta 8 in patient fibroblasts compared with control fibroblasts, in addition to disrupted autophagy pathology.

Conclusions: We report a novel family with autosomal dominant RVM caused by the c.515dupC mutation of the HSPB8 gene, causing a translational frameshift that results in an elongated protein. Understanding the mechanism for the RVM pathology caused by mutated chaperone will permit novel targeted strategies to alter the natural history progression. As next-generation sequencing becomes more available, additional myopathic families will be identified with HSPB8 mutations.

Figure 1. Family pedigree showing autosomal dominant inheritance

The filled upper right quadrant indicates myopathy, and the filled upper left quadrant indicates nonspecific neurologic symptoms. The arrow indicates the proband.

Figure 2. Muscular involvement of the HSPB8 disease

(A) Photograph of the proband showing scapular winging and bilateral wasting of deltoids. (B) T1-weighted MRI of the proband showing severe relatively symmetric multifocal fatty degenerative changes of bilateral lower extremities and (C) proximal lower extremities preferentially affecting the vastus medialis and intermedius together with the adductor magnus and semitendinosus on the thigh level, as well as predominately anterior and lateral compartments more than soleus muscle of the distal legs.

Figure 3. Muscle biopsy shows rimmed vacuoles

(A) Muscle histology of a biopsy of the left vastus lateralis from case 2 shows the presence of rimmed vacuoles (in 3 fibers), adipose replacement, moderate to severe endomysial fibrosis, muscle fiber size variation, numerous atrophic muscle fibers, and increase in central nuclei. (B) Semithin section stained with toluidine blue showing a single fiber with accumulated autophagic vacuoles, corresponding to rimmed vacuolar changes in muscle cryosections.

Figure 4. Sequencing of the HSPB8 gene

(A) Chromatogram from the proband revealing the frameshift mutation c.515dupC p.Pro173fs, which was similarly identified in all affected individuals. (B) The mRNA that results from this mutation has 5 splice variants with size ranging from 27 to 244 aa. Only the first 3 contain an α-crystallin domain and are coding. The red boxes are exons with their size being proportional to their length. The wide colored boxes are introns. Similarly colored introns are identical. The purple introns are upstream open reading frames. The solid black vertical lines indicate validated cap sites at the 5′ end, and the dotted black lines indicate validated polyadenylation sites at the 3′ end. The clear boxes are untranslated regions. (C) Conservation of the sequence of amino acids within HSPB8 across species reveals a very well conserved protein. (D) The predicted sequence of amino acids in the mutant HSPB8 protein (214 amino acids) is longer than its wild-type counterpart (196 amino acids).

Figure 5. Expression of HSPB8, LC3B, p62/SQSTM1, and BAG3 proteins comparison in patients versus controls

(A) Western blot revealed low HSPB8, increased autophagy (LC3, p62) protein expression in patient fibroblasts compared with age- and sex-matched controls. GAPDH was used as a positive loading control. (B) Densitometry analyses of the Western blot results (C) Immunocytochemistry images by confocal microcopy confirmed Western blotting data. Pair 1: patient (P1) and control (C1) fibroblasts; pair 2: P2 and C2; pair 3: P3 and C3.

Figure 6. Expression of HSPB8 protein under heat shock condition

(A) No heat shock condition, (B) heat shock condition. An increase in the amount of HSPB8 protein was present in 3 heated fibroblasts from patients (B1) compared with nonheated fibroblasts (A1). This phenomenon was not observed in control cells (A2 and B2). Pair 1: patient (P1) and control (C1) fibroblasts; pair 2: P2 and C2; pair 3: P3 and C3.