Differential diagnosis of vacuolar myopathies in the NGS era

Abstract

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non‐inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late‐onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr‐caveolinopathy and of limb‐girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.

Short-Communication: Variable Expression of Clinical Symptoms and an Unexpected Finding of Vacuolar Myopathy Related to a Pathogenic Variant in the CACNA1S Gene in a Previous Case Report

“…Based on our patient’s medical history, it was the reason we suspected channelopathy as the main cause of his symptoms. Since there are previous reports of vacuolar myopathy associated with other genetic causes as part of the differential diagnosis, for example, defects of a lysosomal pathway like a glycogen storage disease II or Pompe disease, Danon disease by a mutation in the LAMP2, in an unusual facioscapulohumeral muscular dystrophy (FSHD) phenotype of X-linked myopathy with excessive autophagy (MEAX) caused by a mutation in the VMA21 gene, an essential assembly chaperone of the vacuolar ATPase [ 20 ], we decided to proceed with an extended panel of genes related to these neuromuscular diseases including but not limited to muscular dystrophies, inherited myopathies, mitochondrial disorders, congenital myasthenic syndromes, and rhabdomyolysis. The genetic heterogeneity associated with these conditions can make it difficult to use phenotype as the sole criterion to select a definitive cause.…”

Phenotypical and Myopathological Consequences of Compound Heterozygous Missense and Nonsense Variants in SLC18A3

“…A biopsy of the vastus lateralis muscle was performed for diagnostic purposes. Cryostat sections (6 µm) were processed for routine histological and immunohistochemical staining and histochemical reactions including Oil Red O. Electron microscopy (EM) of ultrathin sections from glutaraldehyde-fixed, resin-embedded muscle was performed using a Philips CM10 transmission electron microscope [ 9 ].…”

Muscle pathology in neuromuscular disorders

“…Vacuolar myopathy is characterized by autophagic vacuoles, which are clearly evident as rimmed vacuoles under a light microscope. 16,17 Rimmed vacuoles appear as empty spaces rimmed by red granules in mGT staining (Fig. 6A), and their ultrastructural findings are typified by clusters of vacuoles containing amorphous, myeloid structures (Fig.…”