Mutation in BAG3 causes severe dominant childhood muscular dystrophy

Abstract

Objective: Myofibrillar myopathies (MFMs) are morphologically distinct but genetically heterogeneous muscular dystrophies in which disintegration of Z disks and then of myofibrils is followed by ectopic accumulation of multiple proteins. Cardiomyopathy, neuropathy, and dominant inheritance are frequent associated features. Mutations in alphaB-crystallin, desmin, myotilin, Zasp, or filamin-C can cause MFMs and were detected in 32 of 85 patients of the Mayo MFM cohort. Bag3, another Z-disk-associated protein, has antiapoptotic properties, and its targeted deletion in mice causes fulminant myopathy with early lethality. We therefore searched for mutations in BAG3 in 53 unrelated MFM patients.

Methods: We searched for mutations in BAG3 by direct sequencing. We analyzed structural changes in muscle by histochemistry, immunocytochemistry, and electron microscopy, examined mobility of the mutant Bag3 by nondenaturing electrophoresis, and searched for abnormal aggregation of the mutant protein in COS-7 (SV-40 transformed monkey kidney fibroblast-7) cells.

Results: We identified a heterozygous p.Pro209Leu mutation in three patients. All presented in childhood, had progressive limb and axial muscle weakness, and experienced development of cardiomyopathy and severe respiratory insufficiency in their teens; two had rigid spines, and one a peripheral neuropathy. Electron microscopy showed disintegration of Z disks, extensive accumulation of granular debris and larger inclusions, and apoptosis of 8% of the nuclei. On nondenaturing electrophoresis of muscle extracts, the Bag3 complex migrated faster in patient than control extracts, and expression of FLAG-labeled mutant and wild-type Bag3 in COS cells showed abnormal aggregation of the mutant protein.

Interpretation: We conclude mutation in Bag3 defines a novel severe autosomal dominant childhood muscular dystrophy.

Fig. 1

(A) Scheme of the genomic structure of BAG3 and the identified mutation. (B) Alignment of amino acid sequences of human, chimpanzee, dog, bovine, rat, mouse, chicken, and zebra fish.

Fig. 2

(A-C): Characteristic histologic findings in trichrome (A), NADH dehydrogenase (B) and Congo red (C) stained sections. (D-J): sections stained trichromatically (D), and immunoreacted for Bag3 (E), αB-crystallin (F), desmin (G), gelsolin (H), NCAM (I), and heat shock protein 27 (J). Note abnormal accumulation of each protein in the structurally abnormal fibers. (K): Modified TUNEL stain reveals apoptotic nuclei in two adjacent fibers. Panels A and B, and panels D-J are nonconsecutive sections from two different series. For panels (A-C), Bars = 100 μm; for panels (D-K), bar in K = 50 μm.

Fig. 3

Electron microscopy. (A) Z-disk (Z) streaming (arrow) and accumulation of small pleomorphic dense structures between the myofibrils (asterisk). (B) More advanced alterations include disintegration and disarray of the myofibrils, aggregation of fragmented and degraded filaments into dense inclusions (X), and accumulation of granular debris. (C) Further destructive changes result in loss of myofibrillar integrity, appearance of dilated vesicles, and aggregation of mitochondria into clusters (m). (D and E) Apoptotic nuclei (n) in a fiber with dilated vesicles (D), and in a highly degenerate fiber (E).

Fig. 4

Nuclear alterations in Patient 1. (A) Ovoid nucleus with prominent nucleolus suggesting increased transcription (arrow) and apoptotic nucleus (X). (B) Large nucleus harboring clumps of heterochromatin. (C) A superficially positioned shrunken (pyknotic) nucleus in a muscle fiber is positioned under an exocytosed pyknotic nucleus. Arrow points to collagen fibrils in the extracellular space. (D) An exocytosed apoptotic nucleus. Arrow points to collagen in the extracellular space. Bars = 2 μm in (A) and (C) and 1 μm in (B) and (D).

Fig. 5

Bag3 immunoblot of native muscle extract of Patient 1 and two normal controls electrophoresed under nondenaturing conditions. Patient displays a faster migrating Bag3 band than controls.

Fig. 6

(A-C): COS-7 cells transfected with FLAG-labeled wild-type (A) and mutant BAG3 (B and C). The nuclei are stained with DAPI. Wild-type Bag3 localizes to a reticular network of fine filaments; mutant Bag3 localizes predominantly to fine discrete granules. Apotome optics, 0.43 μm slice distance. Bar = 20μm for each panel. (D) Frequencies of COS-7 cells harboring reticular, granular, globular and clumped Bag3-positive profiles. Transfection with the mutant construct results in many more cells harboring granular profiles (P < 0.001), and fewer cells with reticular profiles (P < 0.001), than transfection with the wild type construct. Four-hundred and forty-five cells expressing wild-type Bag3 and 470 cells expressing mutant Bag3, were analyzed. Vertical bars indicate standard deviations.