Genetic defects are common in myopathies with tubular aggregates

Abstract

Objective: A group of genes have been reported to be associated with myopathies with tubular aggregates (TAs). Many cases with TAs still lack of genetic clarification. This study aims to explore the genetic background of cases with TAs in order to improve our knowledge of the pathogenesis of these rare pathological structures.

Methods: Thirty-three patients including two family members with biopsy confirmed TAs were collected. Whole-exome sequencing was performed on 31 unrelated index patients and a candidate gene search strategy was conducted. The identified variants were confirmed by Sanger sequencing. The wild-type and the mutant p.Ala11Thr of ALG14 were transfected into human embryonic kidney 293 cells (HEK293), and western blot analysis was performed to quantify protein expression levels.

Results: Eleven index cases (33%) were found to have pathogenic variant or likely pathogenic variants in STIM1, ORAI1, PGAM2, SCN4A, CASQ1 and ALG14. Among them, the c.764A>T (p.Glu255Val) in STIM1 and the c.1333G>C (p.Val445Leu) in SCN4A were novel. Western blot analysis showed that the expression of ALG14 protein was severely reduced in the mutant ALG14 HEK293 cells (p.Ala11Thr) compared with wild type. The ALG14 variants might be associated with TAs in patients with complex multisystem disorders.

Interpretation: This study expands the phenotypic and genotypic spectrums of myopathies with TAs. Our findings further confirm previous hypothesis that genes related with calcium signalling pathway and N-linked glycosylation pathway are the main genetic causes of myopathies with TAs.

Figure 1

Pedigrees of the Family A and Family B. Black symbols represent affected patients; Black arrow points the index patient of the family; Symbols with slash indicate deceased individuals; Numbers inside the symbols indicate multiple individuals with the same gender; Diamond with a question mark indicates that the number of individuals with unknown gender was unclear.

Figure 2

Pathological changes observed in Case 2. Hematoxylin and eosin staining shows variation in fibre size with frequent atrophic fibres and increased connective tissue. Prominent basophilic inclusions are present in many fibres (A); Modified Gomori trichrome staining highlights bright red inclusions (B); The inclusions were stained dark in NADH‐TR (C) and adenylate deaminase preparations (G); The inclusions were immunoreactive for SERCA II (D); Fast myosin (E) and slow myosin (F) immunohistochemistry indicated both type I and type II fibres were affected; Accumulations of double‐walled tubular aggregates were confirmed in electron microscopy (EM) (H). Inclusions of tubular aggregates are indicated by arrows in images A‐C, G and H. Scale bar represents 50 μm in A‐G for histology images; Scale bar in H represents 500 nm in EM

Figure 3

Pathological changes observed in Case 3 and Case 5. Case 3 (A‐D): There were numerous subsarcolemmal and internal basophilic inclusions suggesting TAs in muscle fibres stained using hematoxylin and eosin (A), while these inclusions were stained red in the modified Gomori trichrome preparation (B), and were stained as dark in NADH‐TR and mainly in type II fibres (C); In toluidine blue, the inclusions were in dark blue, with small dark dots in fibres representing lipid droplets (D); Case 5 (E‐H): There was increased variation in fibre size and scattered atrophic fibres which were mostly angular or polygonal in outline. Occasional fibres contain basophilic inclusions suggestive of TAs when stained with hematoxylin and eosin (E, F); TAs are stained red in the modified Gomori trichrome preparation (G), and are darkly stained in NADH‐TR (H). Inclusions of TAs are indicated by arrows in all the images. Scale bar represents 25 μm in A, B, D and F‐H; represents 50 μm in C and E.

Figure 4

Functional analysis of the variant p. Ala11Thr in the ALG14 gene. (A) Western blot of HEK293 cells with wild type and p. Ala11Thr mutant ALG14. Transfection efficiency was verified by co‐transfection of EGFP. (B) The level of ALG14 expression was the mean value of three experiments, and the error bar represents the standard deviation. EGFP, enhanced green fluorescent protein; HEK, human embryonic kidney 293; WT, wild type.