INTRODUCTION: Protein 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 protei
n. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family. RESULTS: We 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 (DeltaG/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 A 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. CONCLUSIONS: Our 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: DeltaG/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, DeltaG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/F domain-lacking DNAJB6 protein.
Harms MB, etal., Ann Neurol. 2012 Mar;71(3):407-16. doi: 10.1002/ana.22683. Epub 2012 Feb 14.
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 lis
t 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.
Nam TS, etal., Neuromuscul Disord. 2015 Nov;25(11):843-51. doi: 10.1016/j.nmd.2015.08.002. Epub 2015 Aug 11.
To identify and characterize genetic mutation in a Korean family with limb-girdle muscular dystrophy 1 (LGMD1), we analyzed in the affected family members clinical features, DNAJB6 by Sanger sequencing, muscle structures by magnetic resonance imaging (MRI), and
functional consequences of the identified mutation using a zebrafish model. The clinical phenotypes along with identification of a novel c.271T > C (p.(Phe91Leu)) mutation in DNAJB6 led to the diagnosis of LGMD1D in the affected family members. This mutation presents unique clinical and radiological features compared with other DNAJB6 mutants. All affected members examined showed reduced pulmonary function, and had nasal voice and dysphagia except the two members who were thirteen and twelve years of age at the time of examination. Muscle phenotypes developed between 8 and 11 years of age and were more severe as compared to previously reported LGMD1D patients with mutant DNAJB6. Patients' MRI scans exhibited early involvement of the lateral head of gastrocnemius, in contrast to its late involvement in reported LGMD1D cases. Functional study using zebrafish embryos demonstrated that p.Phe91Leu elicits more severe muscle defects than the reported p.Phe93Leu and p.Pro96Arg mutations. We conclude that a novel p.(Phe91Leu) mutation in DNAJB6 is associated with severe childhood-onset LGMD1D.
Li S, etal., Hum Mol Genet. 2016 Mar 1;25(5):936-50. doi: 10.1093/hmg/ddv627. Epub 2016 Jan 6.
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 dis
tinct 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.
DNAJB6 is the causative gene for limb-girdle muscular dystrophy 1D (LGMD1D). Four different coding missense mutations, p.F89I, p.F93I, p.F93L, and p.P96R, have been reported in families from Europe, North America and Asia. The previously known mutations cause ma
inly adult-onset proximal muscle weakness with moderate progression and without respiratory involvement. A Finnish family and a British patient have been studied extensively due to a severe muscular dystrophy. The patients had childhood-onset LGMD, loss of ambulation in early adulthood and respiratory involvement; one patient died of respiratory failure aged 32. Two novel mutations, c.271T > A (p.F91I) and c.271T > C (p.F91L), in DNAJB6 were identified by whole exome sequencing as a cause of this severe form of LGMD1D. The results were confirmed by Sanger sequencing. The anti-aggregation effect of the mutant DNAJB6 was investigated in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. Both novel mutant proteins show a significant loss of ability to prevent aggregation.
BACKGROUND & AIMS: The DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) is part of a family of proteins that regulates chaperone activities. One of its isoforms, DNAJB6a, contains a nuclear localization signal and regula
tes beta-catenin signaling during breast cancer development. We investigated the role of DNAJB6 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). METHODS: We performed immunohistochemical analyses of primary ESCC samples and lymph node metastases from a cohort of 160 patients who underwent esophagectomy with no preoperative chemoradiotherapy at Hong Kong Queen Mary Hospital. Data were collected on patient outcomes over a median time of 12.1 +/- 2.9 months. Retrospective survival association analyses were performed. Wild-type and mutant forms of DNAJB6a were overexpressed in cancer cell lines (KYSE510, KYSE 30TSI, KYSE140, and KYSE70TS), which were analyzed in proliferation and immunoblot assays, or injected subcutaneously into nude mice. Levels of DNAJB6 were knocked down in ESCC cell lines (KYSE450 and T.Tn), immortalized normal esophageal epithelial cell lines (NE3 and NE083), and other cells with short hairpin RNAs, or by genome engineering. Bimolecular fluorescence complementation was used to study interactions between proteins in living cells. RESULTS: In primary ESCC samples, patients whose tumors had high nuclear levels of DNAJB6 had longer overall survival times (19.2 +/- 1.8 months; 95% confidence interval [CI], 15.6-22.8 mo) than patients whose tumors had low nuclear levels of DNAJB6 (12.6 +/- 1.4 mo; 95% CI, 9.8-15.4 mo; P = .004, log-rank test). Based on Cox regression analysis, patients whose tumors had high nuclear levels of DNAJB6 had a lower risk of death than patients with low levels (hazard ratio, 0.562; 95% CI, 0.379-0.834; P = .004). Based on log-rank analysis and Cox regression analysis, the combination of the nuclear level of DNAJB6 and the presence of lymph node metastases at diagnosis could be used to stratify patients into groups with good or bad outcomes (P < .0005 for both analyses). There was a negative association between the nuclear level of DNAJB6 and the presence of lymph node metastases (P = .022; Pearson chi(2) test). Cancer cell lines that overexpressed DNAJB6a formed tumors more slowly in nude mice than control cells or cells that expressed a mutant form of DNAJB6a that did not localize to the nucleus. DNAJB6 knockdown in cancer cell lines promoted their growth as xenograft tumors in mice. A motif of histidine, proline, and aspartic acid in the J domain of DNAJB6a was required for its tumor-suppressive effects and signaling via AKT1. Loss of DNAJB6a resulted in up-regulation of AKT signaling in cancer cell lines and immortalized esophageal epithelial cells. Expression of a constitutively active form of AKT1 restored proliferation to tumor cells that overexpressed DNAJB6a, and DNAJB6a formed a complex with AKT1 in living cells. The expression of DNAJB6a reduced the sensitivity of ESCC to AKT inhibitors; the expression level of DNAJB6a affected AKT signaling in multiple cancer cell lines. CONCLUSIONS: Nuclear localization of DNAJB6 is associated with longer survival times of patients with ESCC. DNAJB6a reduces AKT signaling, and DNAJB6 expression in cancer cells reduces their proliferation and growth of xenograft tumors in mice. DNAJB6a might be developed as a biomarker for progression of ESCC.
Reidy M, etal., J Biol Chem. 2016 Feb 19;291(8):4035-47. doi: 10.1074/jbc.M115.700393. Epub 2015 Dec 23.
Human chaperone DnaJB6, an Hsp70 co-chaperone whose defects cause myopathies, protects cells from polyglutamine toxicity and prevents purified polyglutamine and Abeta peptides from forming amyloid. Yeast prions [URE3] and [PSI(+)] propagate as amyloid forms of Ure2 and Sup35 proteins, respectively.
Here we find DnaJB6-protected yeast cells from polyglutamine toxicity and cured yeast of both [URE3] prions and weak variants of [PSI(+)] prions but not strong [PSI(+)] prions. Weak and strong variants of [PSI(+)] differ only in the structural conformation of their amyloid cores. In line with its anti-prion effects, DnaJB6 prevented purified Sup35NM from forming amyloids at 37 degrees C, which produce predominantly weak [PSI(+)] variants when used to infect yeast, but not at 4 degrees C, which produces mostly strong [PSI(+)] variants. Thus, structurally distinct amyloids composed of the same protein were differentially sensitive to the anti-amyloid activity of DnaJB6 both in vitro and in vivo. These findings have important implications for strategies using DnaJB6 as a target for therapy in amyloid disorders.
