The disorder in the two U.S. families was linked originally to 7q36 and classified as LGMD1D¹. Clinical and genetic characterization of the Finnish families then established linkage to the same locus and refined LGMD1D to a 3.4-Mb region containing 12 genes^{3, 4}. Sequencing of the positional candidates RNF32, UBE3C, DNAJB6, and PTPRN2 revealed a c.279C>G (p.Phe93Leu) change in exon 5 of DNAJB6 in all 16 affected patients in the Finnish families FF1–5 (nucleotide and protein numberings based on NM_005494.2 and NP_005485.1, respectively). The mutation was absent from 12 unaffected individuals in these families (Table 1, Supplementary Figs. 1, 2). Sequencing of muscle cDNA from two Finnish patients confirmed that the wild-type and mutant alleles were both expressed (Supplementary Fig. 2). Analysis of DNAJB6 in the two U.S. families revealed a c.267T>A (p.Phe89Ile) mutation in all 29 affected individuals (Table 1), and in two individuals of unknown disease status (below the highest age of onset identified previously in the families). Two discrete DNAJB6 mutations were identified in the Italian families; c.279C>A (IT1; eight patients) and c.277T>C (IT2; four patients), both leading to the same p.Phe93Leu change as the Finnish mutation (Table 1). Six available healthy individuals in IT1 and four in IT2 had no mutations. None of the mutations were found in 404 Finnish, 208 Italian or 430 U.S. control chromosomes. The presence of multiple independent mutations and their segregation with the phenotype in nine families comprises conclusive genetic evidence that DNAJB6 is the LGMD1D causative gene.

DNAJB6 (also known as MRJ) belongs to the J-proteins (also known as the Hsp40 family), a class of co-chaperones characterized by a J-domain. These co-chaperones interact with the chaperones of the HSPA (Hsp70) family, increasing and modulating their activity (recently reviewed by Kampinga & Craig⁶). Some J-proteins, including DNAJB6, also have HSPA-independent functions^{6, 7}.

DNAJB6 is composed of a conserved N-terminal J-domain, a G/F-domain rich in glycine and phenylalanine residues, and a C-terminal domain containing a serine-rich (“SSF-SST”) region^{7, 8} (Supplementary Fig. 2a). The J-domain interacts with the constitutively expressed chaperone HSPA8 (also known as Hsc70 or Hsp73)⁹. The SSF-SST region has been shown to be important for interactions with NFATc3¹⁰ and HDACs⁷, and for DNAJB6 oligomerization⁷. The G/F-domain, harboring the LGMD1D mutations, has been suggested to participate in recognition of partially unfolded client proteins in bacterial DnaJ^11-13 and yeast Sis1¹⁴. Notably, human DNAJB6 has two known isoforms characterized by alternative C-termini¹⁵. The long isoform DNAJB6a (36 kDa) localizes to the nucleus, whereas the short DNAJB6b (27 kDa) is cytoplasmic^{15, 16}.

DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein^{7, 8, 17}, and to participate in autophagic and proteasomal turnover of proteins and organelles^{18, 19}, as well as in regulation of gene expression¹⁰ and cell cycle²⁰. The inhibition of aggregate-induced cytotoxicity may involve myeloid leukemia factor 1 (MLF1), which interacts with DNAJB6 and co-localizes with it in aggregates²¹.

DNAJB6 is expressed in all tissues²² with highest expression in brain⁸, but its expression and localization in skeletal muscle has not been characterized previously. Immunofluorescence (IF) microscopy showed DNAJB6 primarily in Z-disks in both control (not shown) and LGMD1D muscle samples (Fig. 1a). Electron microscopy (EM) of LGMD1D patient muscle revealed Z-disk myofibrillar disintegration (Fig. 1b). Autophagic rimmed-vacuolar degenerative pathology was demonstrated by EM (Fig. 1b, Supplementary Fig. 3), LC3 immunohistochemistry, and Herovici staining (Fig. 1c). IF microscopy of patient muscle revealed DNAJB6 in protein accumulations together with its known ligands MLF1²¹ and HSPA8⁹ (Fig. 1d, Supplementary Fig. 4). Myotilin, desmin, and αB-crystallin, and occasionally filamin—proteins typically aggregating in myofibrillar myopathies—were also observed in these structures. Furthermore, some accumulations stained positive for keratin 18 (KRT18), a reported client protein of DNAJB6⁹ (Fig. 1d, Supplementary Fig. 4). Notably, we did not observe accumulation of thick and thin filament components, such as myosins and actin (not shown). For comparison, we examined muscle tissue from a myofibrillar myopathy patient carrying the p.Ser60Cys myotilin mutation, characterized by aggregation of myotilin and other proteins²³. We found co-localization of DNAJB6, MLF1 and KRT18 with myotilin in these aggregates (Supplementary Fig. 4). In LGMD1D muscle, DNAJB6 appeared more in the periphery of the protein accumulations, in contrast to the more pronounced co-localization seen in myotilinopathy.

To provide further support for the causal involvement of DNAJB6, we investigated its roles for muscle integrity in vivo in zebrafish. Reciprocal BLAST identified in zebrafish a single DNAJB6 ortholog (dnajb6b). We detected its expression by RT-PCR as early as the embryonic 5-somite stage (data not shown). Next, we injected two-cell stage embryos with a splice-blocking morpholino (sb-MO) targeting dnajb6b. Masked scoring of embryos at 48 hours post-fertilization (hpf) showed a reproducible muscle fiber detachment phenotype concomitant with the splice blocking efficiency, as established by RT-PCR (Supplementary Fig. 5a). Detachment of slow fibers from their insertion sites at the vertical myoseptum was evident as early as 2 days post-fertilization (dpf) (Fig. 2), suggesting that the fiber termini in morphants are prone to adhesion failure soon after mechanical load is applied by the onset of strong contraction²⁴. The phenotype was specific; it could be reproduced with a second non-overlapping MO (data not shown), and rescued with co-injection of wild-type human DNAJB6b mRNA (Fig. 2j). These data suggest that loss of function of DNAJB6 leads to muscle integrity defects.

To understand the mechanistic effect of LGMD1D mutations, we introduced the p.Phe93Leu and p.Phe89Ile changes in human transcripts encoding each of the two isoforms, and injected them in zebrafish embryos. In contrast to wild-type DNAJB6, which yielded no appreciable defects, injection of equivalent doses of either p.Phe93Leu or p.Phe89Ile mRNAs phenocopied the loss-of-function effects of the dnajb6b sb-MO (Fig. 2). Strikingly, these phenotypes were generated exclusively upon injection of mutant DNAJB6b; neither mutation, when engineered into DNAJB6a, had any impact (Fig. 2).

All identified LGMD1D mutations map to the G/F-domain of DNAJB6. Both mutated Phe residues are highly conserved in DNAJB6 orthologs, and Phe93 is also conserved in several paralogs (Supplementary Fig. 2). It is unusual that three of the four LGMD1D-causing mutations affect the same codon, leading to the identical p.Phe93Leu change. To determine whether the loss of Phe or the introduction of Leu is more important, we replaced Phe93 with either Ala or Gly. Embryos injected with either p.Phe93Ala or p.Phe93Gly mRNA showed muscle fiber detachment similar to the p.Phe93Leu-induced phenotype (Fig. 2), indicating that the loss of Phe93 is sufficient for the pathogenesis. However, the zebrafish ortholog contains a Gly instead of Phe89, suggesting that some changes might be tolerated in this position; expression of human p.Phe89Gly DNAJB6b in zebrafish embryos did not cause a muscle phenotype (Fig. 2).

Next, we tested whether the LGMD1D mutations could be rescued with wild-type DNAJB6. Co-injection of equimolar amounts of p.Phe93Leu and wild-type mRNA into zebrafish embryos showed enhanced severity of the muscular phenotype: in comparison to 15% of embryos injected with mutant DNAJB6b alone, 42% of co-injected embryos had severe defects in muscle integrity by 2 dpf (P=0.0004; Fig. 3a). A similar effect was observed with the p.Phe89Ile mutation (P=0.0002; Fig. 3a). Taken together, our data indicate that the phenotype is driven by the dysfunction of the cytoplasmic isoform and suggest that the LGMD-causing mutations likely exert a deleterious dominant effect on the wild-type protein. Consistent with this notion, altering the molar ratios of mutant and wild-type mRNA had the expected effects on the phenotype. An excess of mutant to wild-type mRNA induced lethality in embryos, while an excess of wild-type to mutant mRNA gave rise to progressively increased rescue (Supplementary Fig. 5c).

We next turned to in vitro experiments. Based on evidence from the closely related DNAJB8 protein, DNAJB6 has been suggested to form oligomeric complexes⁷. As interfering with the complex formation could explain the dominant effect of the mutations, we studied the oligomerization of wild-type and p.Phe93Leu DNAJB6b in COS-1 cells. In coimmunoprecipitation (CoIP) experiments, V5-tagged DNAJB6b pulled down the untagged construct, demonstrating oligomerization (Supplementary Fig. 6a), and this was unaffected by the p.Phe93Leu mutation. Likewise, in sucrose density gradient centrifugation, the distribution of wild-type and mutant constructs was similar (Supplementary Fig. 6b). Hence, the p.Phe93Leu mutation did not significantly alter the oligomerization properties of DNAJB6.

We next turned to the possibility that LGMD1D mutations might alter DNAJB6 half-life, as we observed that cells transfected with DNAJB6b showed an increase in mutant protein abundance with no appreciable changes in mRNA levels (data not shown). To examine this, we transfected 293FT cells, followed by blocking protein synthesis with cycloheximide (CHX) and quantifying the amounts of remaining protein as a function of time (Fig. 3b, Supplementary Fig. 7). Both p.Phe93Leu and p.Phe89Ile decreased significantly the turnover rate of DNAJB6. To explore whether the increased abundance of mutant DNAJB6 might be proteasome or autophagosome-dependent, we treated DNAJB6-transfected cells either with a proteasome poison (lactacystin) or a lysosome inhibitor (bafilomycin A1). Suppression of the proteasome had no appreciable effects on the clearance of DNAJB6. By contrast, the bafilomycin treatment (in the presence of CHX) led to a significant (40–50%) increase of DNAJB6, suggesting that, at least in part, DNAJB6 is cleared by the autophagosome pathway (Supplementary Fig. 7). This is also consistent with the observation that DNAJB6 abundance was reduced in the presence of both lactacystin and CHX (compared to CHX alone), since suppression of the proteasome has been reported to enhance autophagy^25-27.

The ability of mutant DNAJB6 to oligomerize, the observed increased abundance of mutant protein, and the exacerbation of the zebrafish phenotypes by co-injection of wild-type mRNA suggested that mutant DNAJB6 might affect the entire oligomeric complex. To assay this, we co-transfected cells with mutant and wild-type DNAJB6b. Not only did the mutant protein decrease the turnover of the wild-type protein, but the stability of the mutant itself increased further in the presence of wild-type DNAJB6 (Fig. 3b). These data confirm that mutant DNAJB6 can exert a dominant effect on the wild-type protein.

A possible explanation for the increased abundance of mutant/wild-type protein complexes would be formation of aggregates. However, we did not detect any increased aggregation propensity of the mutants in cell fractionation, sucrose density gradient centrifugation, or IF microscopy of transfected cells (not shown). Hence, primary aggregation of DNAJB6 is unlikely to underlie the pathogenesis of LGMD1D. This is in line with the muscle pathology revealing DNAJB6 in the periphery, rather than in the center, of the protein accumulations.

DNAJB6 has been shown to suppress aggregation of various proteins in cell culture models^{7, 8, 28}. To investigate the effect of the mutations on the anti-aggregation function, we tested the ability of wild-type and mutant DNAJB6b to inhibit aggregation of polyglutamine-containing huntingtin (pEGFP/HD-120Q) in a filter trap assay (FTA). The nuclear DNAJB6a isoform, known to be inefficient towards cytoplasmic huntingtin aggregation⁷, served as negative control. In line with Hageman et al.⁷, wild-type DNAJB6b efficiently suppressed huntingtin aggregation, as demonstrated by a decrease in the aggregate amount and a concomitant increase in SDS-soluble huntingtin (Fig. 4, Supplementary Fig. 8). In contrast, mutant constructs showed significantly impaired anti-aggregation function, although they still retained some activity. This compromised function could impair protein quality control in LGMD1D muscle, ultimately leading to protein accumulation.

Chaperone-assisted selective autophagy (CASA), mediated by a complex containing HSPA8, BAG3, STUB1 (also known as CHIP), and HSPB8 (also known as Hsp22), is important for Z-disk maintenance^{29, 30}. Several pieces of evidence prompted us to investigate the link between DNAJB6 and CASA. First, DNAJB6 is a known co-chaperone of HSPA8⁸. Second, the pathology of LGMD1D muscle suggests defective Z-disk maintenance. Third, both DNAJB6 and the CASA complex localize to the Z-disk. Finally, mutations in BAG3 cause a myofibrillar myopathy with protein accumulations and autophagic pathology³¹. To study the possible association of DNAJB6 with the CASA complex, we performed CoIP studies of endogenous and transfected proteins in COS-1 cells, and observed interaction of DNAJB6b with BAG3, HSPB8, and STUB1 (Fig. 5a, Supplementary Fig. 9). Association of DNAJB6 with the CASA machinery was also supported by in situ proximity ligation assay (PLA) studies on rat muscles (Fig. 5b). None of the interactions seemed to be affected by the p.Phe93Leu mutation (Fig. 5a, Supplementary Fig. 9), indicating that the phenotype is unlikely to be caused by impaired binding of DNAJB6 to the CASA complex.

The interaction with DNAJB6 suggested a possible role for BAG3 in LGMD1D and implied a pathomechanistic link between LGMD1D and the BAG3 myopathy. We therefore asked whether BAG3 could modulate the phenotype caused by mutant DNAJB6b in zebrafish. Surprisingly, while injection of wild-type BAG3 alone caused no observable phenotype, its co-injection with DNAJB6b p.Phe93Leu accentuated dramatically the muscle defect caused by the mutation (P=0.02; Fig. 5c). In addition, while injection of the BAG3 myopathy mutant (p.Pro209Leu)³¹ alone led to a phenotype comparable to DNAJB6 mutants, its co-injection with DNAJB6b p.Phe93Leu did not have an additive effect. These findings strongly suggest that BAG3 plays an active role in mediating the dominant pathogenic effect of DNAJB6b mutations and that it likely lies downstream of DNAJB6.

We have described here the first human mutations in the ubiquitously expressed, multifunctional co-chaperone DNAJB6. This protein has been implicated previously in a variety of degenerative disorders such as Parkinson’s¹⁷ and Huntington’s⁸ diseases, as well as cancer¹⁶. Surprisingly, the identified DNAJB6 mutations cause a tissue- and isoform-specific disease, muscular dystrophy; despite the high expression of DNAJB6 in the brain, there are no indications of neurological involvement^3-5.

The four LGMD1D mutations all affect two highly conserved, closely spaced phenylalanine residues in the G/F-domain. The mutations impair the anti-aggregation activity of DNAJB6b and interfere with the turnover of both mutant and wild-type DNAJB6b. The unexpected finding that addition of wild-type DNAJB6b exacerbates the muscular phenotype induced by each mutation in zebrafish suggests that incorporation of mutant protein to the DNAJB6 oligomer likely confers a dominant toxic function to the entire complex. Moreover, the interaction of BAG3 with DNAJB6, and its aggravating effect on the zebrafish phenotype suggest a direct role for BAG3 in the pathomechanism. The myofibrillar disintegration with protein accumulations in LGMD1D muscle could therefore reflect impaired function of the CASA machinery, leading to insufficient maintenance of sarcomeric structures or defective clearance of misfolded sarcomeric proteins.

The DNAJB6 mutations cause a dominant late onset muscle disease characterized by abnormal protein accumulations and autophagic pathology. Although the mutations are expressed in both nuclear and cytoplasmic DNAJB6, they affect muscle pathology exclusively through the cytoplasmic isoform. This is reminiscent of the mechanism proposed for Lafora progressive myoclonus epilepsy, in which mutations in EPM2A are expressed in both the nucleus and the cytoplasm, but appear to interfere specifically with its cytoplasmic phosphatase activity³². Surprisingly, the literature is largely bereft of other such examples, despite the documented prevalence of alternative splicing³³. Elucidation of the mechanisms by which certain isoforms of mutated ubiquitously expressed proteins can lead to organ-specific effects will be important for understanding genetic diseases and for the development of therapeutical options.

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