The clinical spectrum of CASQ1-related myopathy

Abstract

Objective: To identify and characterize patients with calsequestrin 1 (CASQ1)-related myopathy.

Methods: Patients selected according to histopathologic features underwent CASQ1 genetic screening. CASQ1-mutated patients were clinically evaluated and underwent muscle MRI. Vacuole morphology and vacuolated fiber type were characterized.

Results: Twenty-two CASQ1-mutated patients (12 families) were identified, 21 sharing the previously described founder mutation (p.Asp244Gly) and 1 with the p.Gly103Asp mutation. Patients usually presented in the sixth decade with exercise intolerance and myalgias and later developed mild to moderate, slowly progressive proximal weakness with quadriceps atrophy and scapular winging. Muscle MRI (n = 11) showed a recurrent fibrofatty substitution pattern. Three patients presented subclinical cardiac abnormalities. Muscle histopathology in patients with p.Asp244Gly showed vacuoles in type II fibers appearing empty in hematoxylin-eosin, Gomori, and nicotinamide adenine dinucleotide (NADH) tetrazolium reductase stains but strongly positive for sarcoplasmic reticulum proteins. The muscle histopathology of p.Gly103Asp mutation was different, showing also NADH-positive accumulation consistent with tubular aggregates.

Conclusions: We report the clinical and molecular details of the largest cohort of CASQ1-mutated patients. A possible heart involvement is presented, further expanding the phenotype of the disease. One mutation is common due to a founder effect, but other mutations are possible. Because of a paucity of symptoms, it is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed.

Figure 1. In silico investigation of p.Gly103Asp

(A) Multiple sequence alignment of calsequestrin 1 (CASQ1) ortholog proteins from different mammals. (B) Stability prediction assessed with different tools. The predicted stability change is reported as predicted by the different tools, with a confidence score reported when available. (C) Cartoon representation of human CASQ1. Magenta spheres represent the position of mutations, and green is used for the putative phosphorylation site. Electrostatic change and molecular dynamics simulation are presented in the box. (D) Sequence feature and secondary structure prediction. Gray, purple, and yellow fells represent disorder, α-helix, and β-sheet, respectively. WT = wild type.

Figure 2. Cumulative incidence plot of the onset of symptoms

Muscle weakness, myalgia, or exercise intolerance in 22 calsequestrin 1 (CASQ1)-mutated patients. 95% Confidence intervals are indicated as dashed lines. Asymptomatic patients are marked by a censoring symbol (+) at the age at last evaluation.

Figure 3. Muscle MRIs

(A) Lower limb muscle MRI. T1-weighted (T1W) sequences in 3 patients of different ages. Age-dependent and asymmetric degree of fibrofatty substitution can be observed initially in the distal portion of vastus lateralis, semitendinosus muscle, and gluteus maximus, late spreading to all anterior thigh muscles with relative sparing of the vastus intermedius and to posterior thigh muscles with relative sparing of sartorius and proximal portion of biceps femoris. A severe involvement of gastrocnemius medialis is shown. (B) Scapular and upper limb muscle MRI. T1W sequences in the 2 oldest patients show moderate involvement of triceps brachii.

Figure 4. Histopathology and immunofluorescence in CASQ-1 myopathy

Serial cryosections of p.Asp244Gly (patient 8) and p.Gly103Asp (patient 14) muscle biopsy (all panels original magnification ×20). In patient 8, in hematoxylin-eosin (H&E) optically empty vacuoles almost exclusively in type II fibers, increased fiber size variation, central nuclei, and splitting are shown. Immunofluorescence analysis of sarcoplasmic Ca²⁺ influx/efflux machinery proteins shows reactivity for calsequestrin 1 (CASQ1), ORAI1, SERCA1, and STIM1 at the edge of the vacuoles that appear optically empty with acidic ATPase. In patient 14, vacuoles filled with amorphous material (H&E) corresponding to tubular aggregates are shown. No optically empty vacuoles are detected. The intrafiber vacuoles are strongly immunoreactive for CASQ1, ORAI1, SERCA1, and STIM1 and weakly immunoreactive for SERCA2.

Figure 5. Histopathology in calsequestrin 1 p.Gly103Asp muscle biopsy

Serial cryosections (*) of patient 14 muscle biopsy (all panels original magnification ×20). Several muscle fibers show vacuoles that appear basophilic in hematoxylin-eosin (H&E), stained red in trichrome (TRI), stained dark blue in nicotinamide adenine dinucleotide (NADH), and did not react with succinic-dehydrogenase (SDH), consistent with tubular aggregates.

Figure 6. Electron microscopy of CASQ1-related myopathy in patients carrying the p.Asp244Gly mutation

Scattered myofibrillar lysis area replaced by (B) a progressive accumulation of glycogen granules is shown. (C) Multiple inclusions, partially or entirely membrane bounded, sometimes gathered to constitute a conglomerate mass with a geometric shape in the myofiber sarcoplasm. The inclusions feature variable electron density from a light loosely textured fibrillar matrix to a homogeneous dark-appearing content. (D) Few tubular aggregates can be also detectable (*) in the subsarcolemmal area.