Summary - Tedesco

A important study by Tedesco et al., published on May 2025, that is collaboration of Angelo Poletti and Viriginia Kimonis ' laboratory teams titled “C-terminal HSPB8 frameshift variants define a toxic gain-of-function signature in MFM13”, in the European Journal of Human Genetics, identifies three novel frameshift mutations in HSPB8 associated with Myofibrillar Myopathy 13 with Rimmed Vacuoles (MFM13). This study builds on existing knowledge and offers new insight into how C-terminal elongations of HSPB8 result in toxic protein aggregation, motor impairment, and multisystem involvement.

Published on:

10/17/2025

An important study by Tedesco et al., published on May 2025, that is collaboration of Angelo Poletti and Viriginia Kimonis ' laboratory teams titled “C-terminal HSPB8 frameshift variants define a toxic gain-of-function signature in MFM13”, in the European Journal of Human Genetics, identifies three novel frameshift mutations in HSPB8 associated with Myofibrillar Myopathy 13 with Rimmed Vacuoles (MFM13). This study builds on existing knowledge and offers new insight into how C-terminal elongations of HSPB8 result in toxic protein aggregation, motor impairment, and multisystem involvement.

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🔍 What Are the New Mutations — and How Are They Unique?

The newly identified mutations— p.Q188Rfs*59, p.Y174Qfs*72 and p.P172Lfs*75—all cause elongation of the HSPB8 C-terminal tail by adding aberrant amino acids beyond the normal stop codon. Although these variants arise from different genetic alterations, they all result in the same frameshift, producing an identical C-terminal sequence. However, depending on the mutation’s position within the gene, the frameshift may begin earlier (e.g., p.Y174Qfs*72) or later (e.g., p.Q188Rfs*59), leading to greater or smaller differences between the mutant and wild-type proteins (Fig. 1).

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*Figure 1. From Tedesco et al, 2025 (Figure 3).* A Comparison of the C-terminus of the novel HSPB8 frameshift variants with the HSPB8 WT and the frameshift variants reported in the literature. The common C-terminal extension and variable C-terminal domain of the HSPB8 frameshift variants previously described are in yellow and purple, respectively [1; the C-terminal extension and C-terminal modification of the novel HSPB8 frameshift variants p.Q188Rfs*59, p.Y174Qfs*72 and p.P172Lfs*75 (here reported) and E192Dfs*55 (reported in [2]) are in blue and green, respectively. The underlined amino acids for E192Dfs*55 are not present in any other sequence. B Intrinsic solubility plot of the C-termini of HSPB8 WT and the frameshift variants obtained with CamSol method [3]. C NSC34 cells expressing HSPB8 WT or its mutants p.P173Sfs*43 and p.Q188Rfs*59. HSPB8 is in green, and nuclei are stained with DAPI (blue); scale bar = 20 µm.

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Clinically, individuals carrying these mutations exhibit progressive muscle weakness affecting both proximal and distal muscle groups. Notably, these cases also involve significant respiratory and cardiac complications, features that appear more prominent than in many previously reported MFM13 cases. This suggests a potentially broader and more severe disease spectrum. Muscle biopsies reveal classic signs of myofibrillar myopathy, including rimmed vacuoles and cytoplasmic protein aggregates, further supporting the pathogenicity of these newly defined variants (Fig. 2).

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*Figure 2. From Tedesco et al. 2025 (Figure 2).* A Histopathological findings of deltoid muscle collected before 30 years of age from patient I: (i and ii) H&E staining showing variation in fiber size, fatty replacement, fiber splitting, endomysial fibrosis, presence of vacuoles and multiple internal nuclei. (iii) Myosin fast staining of type 2 fibers. (iv) Gomori Trichrome shows the rimmed vacuoles containing fuchsinophilic material in certain fibers (arrows); (v) desmin staining revealing aggregation; (vi) HSPB8 is diffusely increased in atrophic fibers, (vii) SQSTM1/p62 is positive in rimmed vacuoles, and (viii) strong TDP-43 reactivity is detected in myofibrillar aggregates. B Histopathological findings of quadriceps muscle from patient II: (i and ii) H&E staining displaying variation in fiber size, atrophy, fat infiltration, focal fibrosis, eosinophilic inclusions, and rimmed vacuoles. (iii) Myosin fast staining of type 2 fibers; (iv) Gomori Trichrome and (v) desmin labeling revealing aggregation; (vi) HSPB8 cytoplasmic aggregates in vacuolated and atrophic fibers, (vii) SQSTM1/p62 labeling showing aggregation, and (viii) punctate TDP-43 labeling in vacuolated fibers. C H&E (i) and Gomori Trichrome (ii) staining of patient III left quadriceps muscle in his 30s showing increased fiber size variation, internal nuclei, and myofibrillar deposits.

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Using transient transfection of human cell lines, the researchers expressed these mutant HSPB8 proteins and observed the following:

All three mutant proteins formed large cytoplasmic aggregates, which co-localized with known autophagy markers such as p62 and LC3B, indicating impaired protein clearance.
The mutant HSPB8 sequestered CASA complex partners—BAG3 and HSP70—into aggregates, thereby disrupting their normal chaperone-assisted selective autophagy (CASA) function.
Cells expressing the mutants showed signs of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) pathways, reflecting broader cellular dysfunction.
Even under conditions that typically enhance autophagy (e.g., treatment with autophagy-inducing agents), mutant HSPB8 aggregates persisted, indicating resistance to normal degradation mechanisms.

These findings strongly support a toxic gain-of-function mechanism, whereby the presence of the mutant protein—not merely its absence—actively disrupts cellular homeostasis.

Crucially, the study demonstrates that the newly defined elongated C-terminal tail itself is likely the key driver of toxicity.

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*Figure 3. From Tedesco et al, 2025.* C NSC34 cells expressing HSPB8 WT or its mutants p.P173Sfs*43 and p.Q188Rfs*59. HSPB8 is in green, and nuclei are stained with DAPI (blue); scale bar = 20 µm. D Western blot on NP-40 soluble and insoluble protein fractions of NSC34 transiently transfected with an empty vector (EV), HSPB8 WT or its mutant p.Q188Rfs*59 and p.P173Sfs*43 constructs. Bar graphs report the optical density relative to HSPB8 normalised on the soluble tubulin (TUBA). One-way ANOVA with Tukey’s test was performed (*p < 0.05; ****p < 0.0001); n = 3. HSPB8 was detected using an antibody recognizing the N-terminal region (NTR). E Immunofluorescence on NSC34 transiently transfected with an empty vector (EV), HSPB8 WT or p.Q188Rfs*59 (red) and HSPB8-WT-GFP (left, green), GFP-BAG3 WT (middle, green) or YFP-HSPA1A (right, green). Nuclei are stained with DAPI (blue); scale bar = 20 µm.

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How Do These Findings Build on What We Already Know?

Earlier work, including a 2023 study by Tedesco et al., had already provided experimental evidence that frameshift mutations in the C-terminal region of HSPB8 are pathogenic—primarily due to their tendency to form toxic aggregates and impair the function of the CASA complex, a critical system for maintaining protein homeostasis.

The new 2025 study builds on this foundation by identifying and characterizing newly diagnosed C-terminally elongated variants. Despite originating from different genetic mutations, these variants all lead to a similar, toxic protein behavior—marked by aggregation and disruption of autophagy pathways—highlighting a convergent pathogenic mechanism driven by the altered C-terminal tail.

Clinically, these new variants are associated not only with progressive muscle weakness but also with pronounced respiratory and cardiac involvement. This broader symptom profile suggests that these mutations may define a more severe and systemic form of MFM13 than earlier variants.

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Why This Study Matters

Confirms toxic gain-of-function model via in vitro evidence
Links C-terminal elongation with convergent pathogenic behavior
Expands known phenotypic spectrum, including systemic involvement
Sets the stage for targeted therapies aimed at toxic C-terminal tails or protein clearance

The authors advocate for therapeutic approaches that reduce mutant HSPB8 levels or boost autophagy, echoing the strategy outlined in earlier reviews.

📖 Read the full article here: https://www.nature.com/articles/s41431-025-01868-z

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