DNAJB6 Mutations Research Articles

Mitochondrial abnormalities contribute to muscle weakness in a Dnajb6 deficient zebrafish model.

Mutations in DNAJB6 are a well-established cause of limb girdle muscular dystrophy type D1 (LGMD D1). Patients with LGMD D1 develop progressive muscle weakness with histology showing fibre damage, autophagic vacuoles, and aggregates. Whilst there are many reports of LGMD D1 patients, the role of DNAJB6 in the muscle is still unclear. In this study, we developed a loss of function zebrafish model in order to investigate the role of Dnajb6. Using a double dnajb6a and dnajb6b mutant model, we show that loss of Dnajb6 leads to a late onset muscle weakness. Interestingly, we find that adult fish lacking Dnajb6 do not have autophagy or myofibril defects, however, they do show mitochondrial changes and damage. This study demonstrates that loss of Dnajb6 causes mitochondrial defects and suggests that this contributes to muscle weakness in LGMD D1. These findings expand our knowledge of the role of Dnajb6 in the muscle and provides a model to screen novel therapies for LGMD D1.

Defining clinical endpoints in limb girdle muscular dystrophy: a GRASP-LGMD study

BackgroundThe Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders.Methods/designThe primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9).DiscussionTo the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable.Trial registrationClinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.

DNAJB6 isoform specific knockdown: Therapeutic potential for limb girdle muscular dystrophy D1

Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved invitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as invivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional invivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.

A novel recessive mutation affecting DNAJB6a causes myofibrillar myopathy

Mutations in the DNAJB6 gene have been identified as rare causes of myofibrillar myopathies. However, the underlying pathophysiologica mechanisms remain elusive. DNAJB6 has two known isoforms, including the nuclear isoform DNAJB6a and the cytoplasmic isoform DNAJB6b, which was thought to be the pathogenic isoform. Here, we report a novel recessive mutation c.695_699del (p. Val 232 Gly fs*7) in the DNAJB6 gene, associated with an apparently recessively inherited late onset distal myofibrillar myopathy in a Chinese family. Notably, the novel mutation localizes to exon 9 and uniquely encodes DNAJB6a. We further identified that this mutation decreases the mRNA and protein levels of DNAJB6a and results in an age-dependent recessive toxic effect on skeletal muscle in knock-in mice. Moreover, the mutant DNAJB6a showed a dose-dependent anti-aggregation effect on polyglutamine-containing proteins in vitro. Taking together, these findings reveal the pathogenic role of DNAJB6a insufficiency in myofibrillar myopathies and expand upon the molecular spectrum of DNAJB6 mutations.

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy.

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.

Neuromuscular Diseases Due to Chaperone Mutations: A Review and Some New Results.

Skeletal muscle and the nervous system depend on efficient protein quality control, and they express chaperones and cochaperones at high levels to maintain protein homeostasis. Mutations in many of these proteins cause neuromuscular diseases, myopathies, and hereditary motor and sensorimotor neuropathies. In this review, we cover mutations in DNAJB6, DNAJB2, αB-crystallin (CRYAB, HSPB5), HSPB1, HSPB3, HSPB8, and BAG3, and discuss the molecular mechanisms by which they cause neuromuscular disease. In addition, previously unpublished results are presented, showing downstream effects of BAG3 p.P209L on DNAJB6 turnover and localization.

Mutations in the J domain of DNAJB6 cause dominant distal myopathy

Eight patients from five families with undiagnosed dominant distal myopathy underwent clinical, neurophysiological and muscle biopsy examinations. Molecular genetic studies were performed using targeted sequencing of all known myopathy genes followed by segregation of the identified mutations in the affected families using Sanger sequencing. Two novel mutations in DNAJB6 J domain, c.149C>T (p.A50V) and c.161A>C (p.E54A), were identified as the cause of disease. The muscle involvement with p.A50V was distal calf-predominant, and the p.E54A was more proximo-distal. Histological findings were similar to those previously reported in DNAJB6 myopathy. In line with reported pathogenic mutations in the glycine/phenylalanine (G/F) domain of DNAJB6, both the novel mutations showed reduced anti-aggregation capacity by filter trap assay and TDP-43 disaggregation assays. Modeling of the protein showed close proximity of the mutated residues with the G/F domain. Myopathy-causing mutations in DNAJB6 are not only located in the G/F domain, but also in the J domain. The identified mutations in the J domain cause dominant distal and proximo-distal myopathy, confirming that mutations in DNAJB6 should be considered in distal myopathy cases.

P.136DNAJB6 integrates sarcomere protein homeostasis and myogenic signaling in coordinated skeletal muscle stress response

Mutations in DNAJB6, encoding a HSP40 co-chaperone, cause LGMD1D, an adult onset progressive myopathy with vacuolar and aggregate myopathology. DNAJB6 has two isoforms both of which contain LGMD1D mutation residues, DNAJB6a which localizes to the nucleus and DNAJB6b which diffusely localizes throughout the cell. The function of each isoform in normal muscle, and how disease associated mutations cause myopathy, is unknown. DNAJB6a is a tumor suppressor and chaperones a multiprotein complex that maintains GSK3β in a dephosphorylated, active, state. DNAJB6b localizes to the Z disc in muscle and may mediate LGMD1D pathogenesis. Prior studies indicate DNAJB6 is important for sarcomeric protein homeostasis. We recently found DNAJB6 not only facilitates protein quality control, but also negatively regulates myogenic signaling through its client, GSK3β. In addition, DNAJB6b mutations inhibit myogenic signaling through augmented GSK3β activity, and GSK3β inhibition improves pathology and strength in LGMD1D mice. The mechanism connecting DNAJB6′s role at the sarcomere with myogenic signaling is unclear. Using an in-vivo imaging strategy in skeletal muscle of live mice, we demonstrate DNAJB6b localizes to the Z disk at baseline, but after myofibrillar injury, it accumulates on the M band and within nuclei. GSK3β, DNAJB6′s myogenic signaling client, also localizes diffusely at baseline, but is excluded from the nucleus following myofibrillar injury, where it is known to suppress hypertrophic signaling through NFATc3. These findings suggest DNAJB6 participates in an integrated stress response in skeletal muscle, coordinating sarcomere repair with modulation of myogenic signaling pathways. In contrast, mutant DNAJB6b mislocalizes to myonuclei and the M band at baseline, and in LGMD1D mice, GSK3β persists within myonuclei after injury. These changes in GSK3β and LGMD1D mutant DNAJB6 localization suggest an uncoupling of sarcomere repair and myogenic signaling in LGMD1D.

Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D.

ObjectiveTo understand DNAJB6's function in skeletal muscle and identify therapeutic targets for limb-girdle muscular dystrophy 1D (LGMD1D).MethodsDNAJB6 knockout (KO) myoblasts were generated with Crispr/cas9 technology, and differentially accumulated proteins were identified using stable isotope labeling, followed by quantitative mass spectrometry. Cultured KO myotubes and mouse muscle from DNAJB6b-WT or DNAJB6b-F93L mice were analyzed using histochemistry, immunohistochemistry, and immunoblot. Mouse functional strength measures included forelimb grip strength and inverted wire hang.ResultsDNAJB6 inactivation leads to the accumulation of sarcomeric proteins and hypertrophic myotubes with an enhanced fusion index. The increased fusion in DNAJB6 KO myotubes correlates with diminished glycogen synthase kinase-β (GSK3β) activity. In contrast, LGMD1D mutations in DNAJB6 enhance GSK3β activation and suppress β-catenin and NFAT3c signaling. GSK3β inhibition with lithium chloride improves muscle size and strength in an LGMD1D preclinical mouse model.ConclusionsOur results suggest that DNAJB6 facilitates protein quality control and negatively regulates myogenic signaling. In addition, LGMD1D-associated DNAJB6 mutations inhibit myogenic signaling through augmented GSK3β activity. GSK3β inhibition with lithium chloride may be a therapeutic option in LGMD1D.

LGMD AUTOSOMAL RESSESSIVE AND DOMINANT: P.108Functional characterization of DNAJB6 J-domain mutations

Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominant mutations in DNAJB6. This ubiquitously expressed co-chaperone interacts with the HSPA (Hsp70) chaperones and promotes their function, and also acts as a powerful suppressor or protein aggregation. Until recently, all of the known LGMD1D-causing mutations in DNAJB6 were located relatively close to each other within the glycine/phenylalanine-rich (GF) domain of the protein. This domain, whose function remains poorly understood, links the HSPA-interacting J domain and the substrate-binding C-terminal domain. The mutations impair the anti-aggregation activity of DNAJB6 and confer a dominant toxic effect to the cytoplasmic isoform DNAJB6 isoform (DNAJB6b). Some GF-domain mutations have also been shown to interfere with the turnover of DNAJB6; however, not all mutations have been characterized in this respect. The first LGMD1D patients with missense variants in the J domain of DNAJB6 have now been identified. To evaluate their pathogenicity and to gain further insight into the molecular pathomechanisms of LGMD1D, we performed functional studies on two putative disease-causing J-domain variants, p.Ala50Val and p.Glu54Ala. The nearby, presumably harmless variant p.Ser57Leu served as control. In filter trap assay, DNAJB6b constructs with disease-associated variants showed impaired anti-aggregation activity towards polyQ-huntingtin, supporting the causative role of the variants and highlighting the importance of the anti-aggregation defect in the pathogenesis of LGMD1D. Protein turnover studies indicated a reduced turnover rate for p.Ala50Val but not for p.Glu54Ala, suggesting that impaired turnover may not be a universal feature of pathogenic DNAJB6 variants.

Novel mutations in DNAJB6 cause LGMD1D and distal myopathy in French families.

The aim was to determine the genetic background of unknown muscular dystrophy in five French families. Twelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested invitro. Five patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation. The mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families.

P.73 - Comprehensive genome analysis of Japanese patients with myofibrillar myopathy

Myofibrillar myopathy (MFM) is genetically and phenotypically heterogeneous group of skeletal and cardiac muscle disorders, which is pathologically characterized by focal disorganization of myofibrils and presence of aggregates of myofibrillar protein. However, the molecular pathomechanism is largely unknown. We performed comprehensive genome analysis to elucidate the pathogenesis of MFM. Among 16888 cases whose pathological diagnosis was made in National Center of Neurology and Psychiatry on 1976 through 2016, a total of 250 patients were categorized as MFM. We then performed mutation screening of known causative genes for MFM (DES, CRYAB, MYOT, LDB3, FLNC, BAG3, FHL1, DNAJB6, VCP, TTN) by combined target re-sequencing with Ion PGM. Finally, we performed exome sequencing in samples in which we did not find any mutation by target re- sequencing. We identified a total of 56 candidate mutations in DES (9 mutations), MYOT (1), LDB3 (10), FLNC (6), BAG3 (3), FHL1 (11), DNAJB6 (5), VCP (11) and TTN (0). By a comparison of our data with public mutation databases, 25 of 56 mutations were considered to be novel: six were identified in LDB3, three in FLNC, two in BAG3, five in FHL1, three in DNAJB6, two in VCP. Mutation catalog of MFM genes presented here should provide useful resources not only for elucidating of pathogenesis, and examining relationship between genotypes and phenotypes, but also for constructing the infrastructure of future clinical sequencing.

Myofibrillar myopathy associated with DNAJB6 mutations (P2.143)

Myofibrillar myopathy associated with DNAJB6 mutations (P2.143)

A novel DNAJB6 mutation causes dominantly inherited distal‐onset myopathy and compromises DNAJB6 function

Mutations in the DNAJB6 gene have been identified as a rare cause of dominantly inherited limb-girdle muscular dystrophy or distal-onset myopathy. Exome sequencing was performed to investigate a Taiwanese family with a dominantly inherited distal-onset myopathy. Functional effects of the causal mutation were investigated in vitro. Exome sequencing of the two affected individuals in this family identified a heterozygous mutation, c.287C>T (p.Pro96Leu) in the DNAJB6 gene, which co-segregated with the myopathy within all 12 family members. Notably, this mutation is novel and localizes within the glycine and phenylalanine-rich (G/F) domain and alters an amino acid residue previously reported with a different mutation. Furthermore, immunofluorescence analyses and filter trap assay demonstrated that the c.287C>T (p.Pro96Leu) mutation possessed a dominant negative effect on the anti-aggregation function of DNAJB6 protein. This study expands the molecular spectrum of DNAJB6 mutations and also emphasizes the pathogenic role of DNAJB6 dysfunction in distal-onset myopathy.

New aspects of myofibrillar myopathies.

Myofibrillar myopathies (MFMs) are hereditary muscle disorders characterized by distinct histopathological features. This review provides an overview of recent research with respect to new disease genes, clinical phenotypes, insights into pathomechanisms and therapeutic strategies. Beyond the known disease genes DES, FLNC, MYOT, CRYAB, ZASP, BAG3, FHL1 and TTN, mutations in PLEC, ACTA1, HSPB8 and DNAJB6 have also been associated with a MFM phenotype. Proteomic analysis revealed new information about the composition of protein aggregates in myotilinopathy and identified a new diagnostic marker. New animal models mirror central aspects of MFM pathology and novel therapeutic strategies for treatment of MFM were evaluated in cell and animal models. MFMs are an expanding and numerically significant group of protein aggregate diseases with marked clinical and genetic heterogeneity. Though no specific therapy is currently available, the generation of patient-mimicking cell and animal models now paves the way for the preclinical evaluation of novel therapeutic strategies.

P.11 - A novel DNAJB6 mutation causing variable phenotypic expression: From distal myopathy to limb girdle muscular dystrophy

P.11 - A novel DNAJB6 mutation causing variable phenotypic expression: From distal myopathy to limb girdle muscular dystrophy

Genetic interaction of hnRNPA2B1 and DNAJB6 in a Drosophila model of multisystem proteinopathy.

Adult-onset inherited myopathies with similar pathological features, including hereditary inclusion body myopathy (hIBM) and limb-girdle muscular dystrophy (LGMD), are a genetically heterogeneous group of muscle diseases. It is unclear whether these inherited myopathies initiated by mutations in distinct classes of genes are etiologically related. Here, we exploit a genetic model system to establish a mechanistic link between diseases caused by mutations in two distinct genes, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila melanogaster homologs of human hnRNPA2B1 and DNAJB6, respectively. We introduced disease-homologous mutations to Hrb98DE, thus capturing mutation-dependent phenotypes in a genetically tractable model system. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins (RBPs), suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover, wild-type, but not disease-associated, mutant forms of MRJ interacted with RBPs after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interactions between disease-linked RBPs and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.

Myofibrillar disruption and RNA-binding protein aggregation in a mouse model of limb-girdle muscular dystrophy 1D.

Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominantly inherited missense mutations in DNAJB6, an Hsp40 co-chaperone. LGMD1D muscle has rimmed vacuoles and inclusion bodies containing DNAJB6, Z-disc proteins and TDP-43. DNAJB6 is expressed as two isoforms; DNAJB6a and DNAJB6b. Both isoforms contain LGMD1D mutant residues and are expressed in human muscle. To identify which mutant isoform confers disease pathogenesis and generate a mouse model of LGMD1D, we evaluated DNAJB6 expression and localization in skeletal muscle as well as generating DNAJB6 isoform specific expressing transgenic mice. DNAJB6a localized to myonuclei while DNAJB6b was sarcoplasmic. LGMD1D mutations in DNAJB6a or DNAJB6b did not alter this localization in mouse muscle. Transgenic mice expressing the LGMD1D mutant, F93L, in DNAJB6b under a muscle-specific promoter became weak, had early lethality and developed muscle pathology consistent with myopathy after 2 months; whereas mice expressing the same F93L mutation in DNAJB6a or overexpressing DNAJB6a or DNAJB6b wild-type transgenes remained unaffected after 1 year. DNAJB6b localized to the Z-disc and DNAJB6b-F93L expressing mouse muscle had myofibrillar disorganization and desmin inclusions. Consistent with DNAJB6 dysfunction, keratin 8/18, a DNAJB6 client also accumulated in DNAJB6b-F93L expressing mouse muscle. The RNA-binding proteins hnRNPA1 and hnRNPA2/B1 accumulated and co-localized with DNAJB6 at sarcoplasmic stress granules suggesting that these proteins maybe novel DNAJB6b clients. Similarly, hnRNPA1 and hnRNPA2/B1 formed sarcoplasmic aggregates in patients with LGMD1D. Our data support that LGMD1D mutations in DNAJB6 disrupt its sarcoplasmic function suggesting a role for DNAJB6b in Z-disc organization and stress granule kinetics.

G.P.173 - A novel mutation in DNAJB6 causes LGMD1D in two French families

Mutations in the co-chaperone DNAJB6 is the cause of LGMD1D (OMIM: #603511). To date, six different protein level changes have been reported (F89I, F91I, F93I, F93L, P96R, and F100I). Interestingly, all mutations are located in the G/F-rich domain of DNAJB6. The various mutations are found in families of European, American and Asian origin. We here report a novel mutation found in two French families that is also located to the G/F-domain of DNAJB6. Clinical symptoms presented between 35 and 50 years of age with proximal lower limb weakness and difficulty rising from chairs. The pathology is congruent with previously reported LGMD1D mutations with progressive loss of muscle fibers, rimmed vacuoles and cytoplasmic protein aggregates that are observed in muscle biopsies from affected muscles. In a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin, which is a model for an aggregation-prone protein, the mutant protein shows significant loss of anti-aggregation capacity compared to the wild-type. DNAJB6 is part of the larger chaperonal complex system handling chaperone mediated selective autophagy. Besides the decreased anti-aggregation function of the new mutant, the functions of the whole complex as previously reported are impaired in a dominant fashion by mutant DNAJB6.

Complete loss of the DNAJB6 G/F domain and novel missense mutations cause distal-onset DNAJB6 myopathy.

IntroductionProtein aggregation is a common cause of neuropathology. The protein aggregation myopathy Limb-Girdle Muscular Dystrophy 1D (LGMD1D) is caused by mutations of amino acids Phe89 or Phe93 of DNAJB6, a co-chaperone of the HSP70 anti-aggregation protein. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family.ResultsWe detail the mutational, neuropathological, neurophysiological, neurological and radiological features of five new DNAJB6-myopathy families. One has the known Phe93Leu mutation and classic late-onset slowly progressive LGMD1D. Two have different mutations of Phe91 causing a variant childhood-onset severe limb-girdle myopathy. One has a Phe100Val mutation and distal-onset myopathy, unique early bulbar involvement, and a gender-modified wide age-of-onset range. The last has childhood-onset severe distal-onset myopathy and the first non-missense DNAJB6 mutation, c.346 + 5G > A, causing a splicing defect that entirely eliminates DNAJB6’s G/F domain (ΔG/F), the domain that harbours all other mutations. Clinical and imaging examinations reveal that muscles considered uninvolved in DNAJB6-myopathy, e.g. lateral gastrocnemii, are affected in our patients with new mutations. Mutational modelling based on the known structure of the bacterial DNAJ2 protein indicates that all past and present mutated residues cluster within 15 Å in the G/F domain and all disturb the interface of this domain with the protein’s J domain that confers the interaction with HSP70.ConclusionsOur patients expand the phenotypic spectrum of DNAJB6-myopathy and allow tentative genotype-phenotype specifications. Combining with previous studies, the clinical severity spectrum is as follows: ΔG/F and Phe91 mutations, most severe; Phe100, Pro96, Phe89 mutations, intermediate; and Phe93, least severe. As it stands presently, proximal G/F domain mutations (Phe89, Phe91, Phe93) cause proximal limb-girdle myopathy, while distal G/F mutations (Pro96, Phe100) cause distal-onset myopathy. While all mutations affect the G/F–J interaction, each likely does so in different unknown extents or ways. One mutation, ΔG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/F domain-lacking DNAJB6 protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0224-0) contains supplementary material, which is available to authorized users.