PMC9259785_01
(A,B) Muscle MRI of lower limbs showed fatty infiltration and muscle atrophy of proximal and distal lower limb muscles, with the most prominent involvement in posterior muscles. (A) Thigh level. The biceps femoris (long head), semitendinosus, vastus lateralis, gracilis, and sartorius muscle are mildly to moderately affected. The rectus femoris is the best spared muscle. (B) Calf level. The gastrocnemius (medial head and lateral head), peroneus (fibularis) brevis, and peroneus (fibularis) longus muscle are moderately affected.
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F1
MRI of the muscles in the lower limbs showed fatty infiltration and muscle atrophy of the lower limb muscles, most prominent involvement in posterior muscles (Figure 1).
(A-F) Serial sections from the patient's left biceps brachii muscle on light microscopy: (A) HE and (B) MGT staining revealed mildly proliferated connective tissue and fiber size variation with atrophic fibers. Rimmed vacuoles were found in a portion of the fibers. Immunohistochemistry showed positive reactivity for (C) TDP-43, (D) p62, and (E) LAMP2 in the rimmed vacuoles. (F) LC3 reactivity was absent or less abundant in the rimmed vacuolar regions. (G) Desmin reactivity showed moderate or strong expression in rimmed vacuolated fibers. (H,I) Ultrastructural changes on electron microscopy: (H) myelin bodies and (I) destroyed myofibrillar structure replaced by loop debris materials were found on transverse sections.
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On light microscopy, HE and MGT staining (Figure 2A,B) revealed mildly proliferated connective tissue and adipose tissue in the perimysium and endomysium and fiber size variation with atrophic fibers. On immunohistochemistry, the rimmed vacuoles showed positive reactivity for TDP-43, p62, and LAMP2 (Figure 2C-E), whereas components of LC3 reactivity were absent or less abundant in the rimmed vacuolar regions (Figure 2F), indicating impaired autophagic flux and defect degradation. Desmin reactivity showed moderate or strong expression in rimmed vacuolated fibers (Figure 2G). On electron microscopy, myelin bodies were found (Figure 2H), and myofibrillar structure was destroyed and replaced by loop debris materials in some regions (Figure 2I).
(A-C) The results of Sanger sequencing of the patient and her parents. (A) A heterozygous mutation c.236-1_240delGGTGGA (p.G79Efs*4) of the DNAJB6 gene was identified, and the mutational site of the patient was confirmed by Sanger sequencing. Sanger verification of her father (B) and mother (C) showed that this site was normal. (D). DNAJB6 transcript sequencing: Gel electrophoresis of the patient showed three different size bands, namely, 547, 493, and 436 bp. The 547 bp band is of the same size as the control one and is a wild-type transcript. By sequencing, the 493 bp band refers to 54-nucleotide deletion at the beginning of exon 5, and the 436 bp band refers to the whole 111-nucleotide delete of exon5, which encodes the G/F domain. (E,F) I-TASSER software was used to develop a suitable model to simulate the effect of the mutation region; the 3D protein modeling predicted that p.79_96del mutation would result in the loss of an alpha-helix (E) and p.79_115del mutation would result in the loss of two alpha-helices (F).
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The mutational site was confirmed by Sanger sequencing, and this site was normal in her parents (Figure 3A-C). Gel electrophoresis of the patient showed three different size bands, namely, 547, 493, and 436 bp (Figure 3D). By sequencing, the 493 bp band refers to 54-nucleotide deletion at the beginning of exon 5, and the 436 bp band refers to the whole 111-nucleotide deletion of exon 5, which encodes the G/F domain (Figure 3D). The 3D protein modeling predicted that p.79_96del mutation would result in the loss of an alpha-helix (Figure 3E) and p.79_115del mutation would result in the loss of two alpha-helices (Figure 3F).