A novel recessive mutation affecting DNAJB6a causes myofibrillar myopathy

Figures

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  Fig. 1. Schematic representation of the DNAJB6 gene exons, DNAJB6 protein isoforms and the mutation. a Sequence comparison of the wildtype DNAJB6a, DNAJB6b and mutant DNAJB6a in human. b Sequence comparison of the wildtype DNAJB6a, DNAJB6b and mutant DNAJB6a in mouse. Note that the alternatively spliced C-terminal part of the “a” or “b” region is identified as the marker of the DNAJB6 isoform

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  Fig. 2. Clinical, histopathological and molecular features of the patient with the DNAJB6 mutation. a The pedigree displayed co-segregation of the p.V232Gfs*7 homozygosis mutation (changes in the genotype labeled in red) with distal-onset myopathy. The arrow indicates the proband (individual II-5). Square: male; circle: female; open symbol: unaffected; filled symbol: affected; symbol with a diagonal line: deceased. b Representative chromatogram of the forward sequencing reaction in the proband displayed a novel mutation in DNAJB6 genomic DNA (c.695_699del; p.V232Gfs*7) (the mutation of genome and amino acids labeled in red arrow and dotted frame). c Patient II5: distal lower limb atrophy (white arrows) and bilateral foot drop (black arrows). d MRI revealed generalized fatty replacement in the distal lower legs, and vastus, gracilis, and semitendinosus muscles (black arrows). Substantial muscle alterations with fatty infiltration were observed in paraspinal, infraspinatus, and intercostal muscles (white arrows). e Histochemical analysis of the vastus lateralis muscle biopsy demonstrated the presence of increased connective tissue, pathological variation of fiber diameter, pyknotic nuclear…

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  Fig. 3. Generation and validation of m-DNAJB6 c.698_702del KI mice. a Schematic representation of the KI mouse DNAJB6 allele displaying the c.698_702del mutation flanked by two homology arms. b Sequencing of the m-DNAJB6 gene demonstrating the presence of the mutation in a single allele of a homozygous (HOM) and wild-type (WT) animal. c-f RT-PCR analysis of mouse-wild type DNAJB6a (wt m-DNAJB6a), mouse-mutant DNAJB6a (mut m-DNAJB6a), mouse-DNAJB6b (m-DNAJB6b) and mouse-total DNAJB6 (total m-DNAJB6a) in the gastrocnemius muscle of HOM or heterozygous (HET) mice and WT littermates. Values are expressed as mean ± SEM (n = 5–13 per genotype). g Western blot analysis showing that the protein lysate from WT exclusively produced a wt m-DNAJB6a band, while HOM or HET mice produced an mutant m-dnajb6a band specifically (arrow). GAPDH was used as a loading control. h-j Quantification of relative intensities of proteins shown in g. Data represent mean ± SEM of 3 independent experiments (n = 3). P value = * < 0.05, ** < 0.01

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  Fig. 4. Assessment of motor function in the HOM and WT mice. a Body weight curves showing changes in body weight over 18 months for HOM and WT mice. Shown are mean ± SEM (n = 12–15) b Hanging test showing “minimal holding impulse” (body mass x hang time), in seconds (s) x gram (g), in 3, 6, 9, 12, 18-month old HOM mice and WT littermates. c Mean score in grip strength over time in HOM mice and WT littermates. d Treadmill experiments shown by duration of running, in seconds (s), before showing signs of exhaustion in HOM mice and WT littermates. e Rotarod performance showing mean duration, in seconds (s), spent on the accelerating rotating rod at 3, 6, 9, 12,18-month old HOM and WT mice. Values represent mean ± SEM (n = 12–15). P value = * < 0.05, ** < 0.01

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  Fig. 5. Tibialis anterior muscle histopathology of HOM and WT mice. a–c Representative HE (d), SDH (e) and COX (f) staining of tibialis anterior (TA) muscle from 3,12,18-month-old WT and HOM mice. Scale Bar = 100 µm. d Electron microscopy images of the TA muscle of 3-month-old HOM mice showed basically normal mitochondria regularly positioned adjacent to myofibrillar Z-discs (1). 12-month-old HOM mice displayed vacuolated (white arrow, 2) and enlarged mitochondria (white asterisk, 2) in the intermyofibrillar space, focal accumulations of mitochondria in the subsarcolemmal region (ma, 3), some of them undergoing mitophagy (white arrowheads, 3). 18-month-old HOM mice displayed severe myofiber disruption (black arrowheads, 4) and vacuolated mitochondria (black asterisk, 4). Scale bars = 0.5 µm (1, 3, 4), 1.0 µm (2)

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  Fig. 6. Changes in fiber type composition of tibialis anterior and gastrocnemius muscles in 12-month-old HOM and WT mice. a, b Representative transverse sections of tibialis anterior (TA) (a) and gastrocnemius (GSN) (b) muscles from 12-month-old WT and HOM mice immunostained with fiber type-specific myosin heavy chain antibodies. Scale bar: 200 µm. Note the decrease in proportion of type IIA fibers and increase in type IIB fibers in the TA from HOM mice (a), and the decrease in type I fibers in parallel with an increase in type IIB fibers in the GSN from HOM mice (b). c, d Quantified fiber type distribution by percent of total in individual fiber types. Values are expressed as mean ± SEM (n = 3). P value = * < 0.05, ** < 0.01

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  Fig. 7. Aggregation of myofibril components in skeletal muscle tissue of 18-month-old HOM and WT mice. a DNAJB6, desmin, dysferlin, TDP-43, LC3B and p62 staining displayed altered subcellular distribution in the HOM mice. Scale bar = 20 µm. b Percentage of myofibers with TDP-43, LC3B and p62 aggregation in 18-month-old HOM and WT mice. c Western blot showing TDP-43 and LC3B in muscle lysates of 18-month-old HOM and WT mice. GAPDH was used as a loading control. d Quantification of relative intensities of proteins shown in c. Data represent means ± SEM of 3 independent experiments (n = 3). P value = * < 0.05, ** < 0.01

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  Fig. 8. Effects of the c.695_699del recessive mutation in DNAJB6 on its mRNA and protein stabilities in cells. a Lysates from HEK293 cell extracts transfected with 500 ng wild type M2-fused DNAJB6a (WT), 500 ng or 1500 ng M2-fused mutant DNAJB6a (500 ng mut, 1500 ng mut) plasmids, followed by immunoblot analysis with antibodies against M2 and β-actin. b Quantification of data in a. Data from three experiments (mean ± SEM). c RT-PCR analysis of WT or mutant DNAJB6a constructs transferred in HEK293 cells after actinomycin D (5 ug/ml) treatment for the indicated times points. d Western blot analysis of M2-fused WT or mutant DNAJB6a protein in HEK293 cells followed the treatment of cycloheximide (10 ug/ml) for the indicated time points. e Quantification of data in d. Data from three experiments (mean ± SEM). P value = * < 0.05, ** < 0.01. N.S. = not significant

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  Fig. 9. Effects of the c.695_699del recessive mutation in DNAJB6 on its ability to prevent aggregation of polyQ in cells. a Representative immunofluorescent images of HEK293 cells cotransfected with HttEx1-(Q)74-hrGFP and 500 ng empty vector as control (Con), 500 ng wild type M2-fused DNAJB6a (WT), 200 ng, 500 ng or 1500 ng mutant M2-fused DNAJB6a (200 ng mut, 500 ng mut, 1500 ng mut) plasmids respectively for 48 h. Scale bar = 50 µm. b Western blot showing Lysates from cells treated as in a. with antibodies against GFP, M2, and β-actin. c Quantitative analysis of a, expressing the proportion of cells containing inclusions (each scored by at least three independent observations and then averaged). d Filter-trap assay. Cell lysates in a. were loaded onto cellulose acetate membranes and probed with an anti-GFP antibody to detect aggregation of HttEx1-(Q) 74-hrGFP. e Quantification of data in d. Data from three experiments (mean ± SEM). P value = * < 0.05, ** < 0.01. N.S. = not significant