case_images: PMC10944937_01
case_images: Histological findings in muscle biopsy. H&E stain in our patient (A) showed a great variability in fiber size. Several fibers showed the presence of cytoplasmic and subsarcolemmal optically empty vacuoles, sometimes of conspicuous size (arrows). (B) H&E stain in age-matched healthy control. (C) MGT stain in our patient showed the presence of intracytoplasmic fuchsinophilic granulations (asterisks). (D) MGT stain in age-matched healthy control. (E) ATPase pH 9.4 in our patient and (F) in age-matched control. Scale bar 10 mum.
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case_images: PMC10944937_01_fneur-15-1340693-g001.jpg
case_images: fig1
case_images: The patient is the second-born child to non-consanguineous healthy parents of Italian origins (Supplementary Figure 1). Several fibers showed the presence of cytoplasmic and subsarcolemmal optically empty vacuoles, sometimes of conspicuous size, and intracytoplasmic fuchsinophilic granulations (Figure 1).
case_images: Histological findings in muscle biopsy. Toluidine blue stained semithin section in our patient (A) showed cytoplasmic and subsarcolemmal pale staining storage material (arrows). (B) Toluidine blue stained semithin section in age-matched healthy control. (C) PAS stain in our patient showed increased subsarcolemmal glycogen content (asterisks). (D) PAS stain in age-matched healthy control. Scale bar 10 mum.
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case_images: PMC10944937_01_fneur-15-1340693-g002.jpg
case_images: fig2
case_images: Analysis on semithin sections showed that the vacuoles were PAS positive, indicating an increased glycogen content (Figure 2), and suggesting a glycogen storage disease; in fact, the histopathological features resembled branching or debranching enzyme deficiency (Autosomal Recessive Glycogen Storage Disease type 4 or 3, respectively).
case_images: Ultrastructural analyses of the muscle sample by electron microscopy. (A) Glycogen granules accumulated among the myofibrils and initial thickening of Z line (arrows). (B,C) Large glycogen collections localized at the subsarcolemmal level and at intracytoplasmic sites, several nemaline rods of variable size and shape (arrows), myofibrillar disorganization. (D) High magnification of a nemaline rod and glycogen granules. (E,F) Fibers with areas of compartmentalization. (E) In the left area the muscle fiber contains several nemaline rods. (F) In the upper area the muscle fiber is completely replaced by glycogen. Scale bars (A-C): 0.8 mum, (D): 0.5 mum. (E,F): 3.3 mum. (A,B,F) Asterisks indicate large glycogen collections.
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case_images: PMC10944937_01_fneur-15-1340693-g003.jpg
case_images: fig3
case_images: The subsequent ultrastructural analyses by electron microscopy confirmed the presence of extensive glycogen collections, leading to significant alterations in muscle architecture (Figure 3A). In addition, several cytoplasmic rods of variable size were found in some muscle fibers (Figures 3B-D). Fibers with area of compartmentalization for nemaline rods (Figure 3E) and glycogen granules (Figure 3F) were also observed.
case_images: Genetic studies in our patient. (A) Pedigree of the family. Black symbol indicates the affected patient. (B) Sequence electropherograms showing the presence of the heterozygous c.965 T > A ACTA1 variant in the patient but not in his parents suggesting de novo occurrence of the variant. (C) Evolutionary conservation of the affected residue across species. (D) Diagram showing the structure of ACTA1 gene and protein. Dominant and recessive variants are indicated above and under the diagram, respectively. "!" symbol indicates a variant in which de novo occurrence was suspected or demonstrated; red color indicates variant associated with intranuclear rod myopathy (IRM); italic style is used for variant associated with fiber type disproportion, the underlined style is used for variants associated with muscle description of homogeneous filamentous inclusions containing actin; the "^" symbol was used to indicate variants found in patients with muscle fibers presenting core regions with no contractile material or mitochondria. Dashed lines indicate the presence of a large genomic deletion. (E-G) Distribution of ACTA1 variants based on their exonic location (E), inheritance pattern (F) and mutational types (G).
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case_images: PMC10944937_01_fneur-15-1340693-g004.jpg
case_images: fig4
case_images: T > A p. (Leu322Gln) variant in the ACTA1 gene (NM_001100.3) (Figure 4A). The absence of the variant in Patient's parents supported the hypothesis of its de novo origin (Figure 4B). The variant was not found in available population databases (GnomAD MAF:0), and it is currently classified as likely pathogenic, according to the ACMG guidelines (criteria PP3-strong, PM1 and PM2). All the interrogated in silico prediction tools unanimously assigned a pathogenic behavior to this novel variant, which would affect an evolutionary conserved residue (Figure 4C) located in the large domain of actin, subdomain 3 (amino acids 270-337) (Figure 4D).
case_images: PMC10944937_02