Exome sequencing reveals DNAJB6 mutations in dominantly-inherited myopathy

Abstract

Objective: To identify the causative gene in an autosomal dominant limb-girdle muscular dystrophy (LGMD) with skeletal muscle vacuoles.

Methods: Exome sequencing was used to identify candidate mutations in the studied pedigree. Genome-wide linkage was then used to narrow the list of candidates to a single disease-associated mutation. Additional pedigrees with dominant or sporadic myopathy were screened for mutations in the same gene (DNAJB6) using exome sequencing. Skeletal muscle from affected patients was evaluated with histochemistry and immunohistochemical stains for dystrophy-related proteins, SMI-31, TDP43, and DNAJB6.

Results: Exome analysis in 3 affected individuals from a family with dominant LGMD and vacuolar pathology identified novel candidate mutations in 22 genes. Linkage analysis excluded all variants except a Phe93Leu mutation in the G/F domain of the DNAJB6 gene, which resides within the LGMD locus at 7q36. Analysis of exome sequencing data from other pedigrees with dominant myopathy identified a second G/F domain mutation (Pro96Arg) in DNAJB6. Affected muscle showed mild dystrophic changes, vacuoles, and abnormal aggregation of proteins, including TDP-43 and DNAJB6 itself.

Interpretation: Mutations within the G/F domain of DNAJB6 are a novel cause of dominantly-inherited myopathy. DNAJB6 is a member of the HSP40/DNAJ family of molecular co-chaperones tasked with protecting client proteins from irreversible aggregation during protein synthesis or during times of cellular stress. The abnormal accumulation of several proteins in patient muscle, including DNAJB6 itself, suggest that DNAJB6 function is compromised by the identified G/F domain mutations.

Figure 1. Exome analysis of families with dominantly-inherited myopathy

(A) Pedigree structures for dominant myopathy Families 1 and 2, with filled symbols for affected individuals. Subjects indicated by arrowheads were used for whole-exome sequencing. Linkage analysis used individuals indicated by “*”. Genotypes for the c.277T>C mutation were determined by direct sequencing and are indicated by “+” (mutation carrier) or “−” (non-carrier). (B) Sequence traces confirming the c.277T>C (p.Phe93Leu) and c.287C>G (p.Pro96Arg) variants. (C) Alignment of the DNAJB6 protein showing complete conservation of the mutated residues through D. melanogaster, with a domain diagram of the DNAJB6 protein below showing the position of the dominant myopathy mutations in the G/F domain, the highly conserved J domain and the C-terminal domain. (D) Alignment of human DNAJB6 with S. cerevisiae orthologues performed using ClustalW2 show that the mutations identified in human myopathies overlap with the Sis1 domain required for yeast prion propagation.

Figure 2. Pathologic characterization of skeletal muscle tissue from a subject with DNAJB6 mutation

Clinically affected deltoid tissue from subject II:3 of family 1 (Phe93Leu mutation) was processed and histochemically stained with (A) hematoxylin and eosin or (B) congo red as previously described . Additional immunohistochemistry with (C) anti-SMI-31 (brown), (D and E) anti-DNAJB6 (red) and (F) dual immunofluorescence with DNAJB6 (red) and TDP-43 (green). Anti-DNAJB6 (red) immunostaining was also performed in (G) normal control and (H) sporadic inclusion body myositis patient muscle tissue. DAPI blue nuclear stain is present in (D–H). Scale bar is 100uM.