Mark Screen

Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy

Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago, but its genetic cause has remained elusive. Here we studied nine LGMD-affected families from Finland, the United States and Italy and identified four dominant missense mutations leading to p.Phe93Leu or p.Phe89Ile changes in the ubiquitously expressed co-chaperone DNAJB6. Functional testing in vivo showed that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. Further, we show that DNAJB6 interacts with members of the CASA complex, including the myofibrillar myopathy–causing protein BAG3. Our data identify the genetic cause of LGMD1D, suggest that its pathogenesis is mediated by defective chaperone function and highlight how mutations in a ubiquitously expressed gene can exert effects in a tissue-, isoform- and cellular compartment–specific manner.

Welander distal myopathy is caused by a mutation in the RNA-binding protein TIA1

A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy.

Atypical phenotypes in titinopathies explained by second titin mutations

Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families.

Altered expression and splicing of Ca2+ metabolism genes in myotonic dystrophies DM1 and DM2

A. Vihola, M. Sirito, L. L. Bachinski, O. Raheem, M. Screen, T. Suominen, R. Krahe and B. Udd (2013) Neuropathology and Applied Neurobiology39, 390–405

Gene expression profiling in tibial muscular dystrophy reveals unfolded protein response and altered autophagy.

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCPs accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.

Abnormal Splicing of NEDD4 in Myotonic Dystrophy Type 2: Possible Link to Statin Adverse Reactions

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2.

P.3.13 Gene expression profiling in Welander distal myopathy

Welander distal myopathy (WDM) is a late onset disease caused by a mutation in the C-terminal region of the TIA1 gene. The secondary molecular events resulting from the causative TIA1 mutations to the preterm death of muscle cells in WDM are mostly unknown. In order to identify downstream pathogenetic mechanisms we explored WDM biopsies genetic profile by expression profiling. We compared the changes to the expression profile of the phenotypically overlapping tibial muscular dystrophy (TMD). Six WDM patient biopsies and five healthy control muscle biopsies were used and the expression data was analyzed using Ingenuity Pathway Analysis. We identified biochemical and genetic pathway changes distinctive to WDM, such as 14-3-3 mediated apoptosis, cleavage and polyadenylation of pre-mRNA, protein trafficking and transport, and oxidative phosphorylation pathway changes. We also identified shared changes with TMD that result in the similarity seen in both these distal myopathies, such as SAP-JNK apoptosis, P70S6 mTOR signalling, mitochondrial dysfunction, and protein ubiquitination pathway changes. TIA1 is a key component of stress granules, a mechanism used to protect other cellular mechanisms during stress. The unique oxidative phosphorylation changes we identified by expression profiling may be associated with increased oxidative stress in WDM. TIA1 is also involved in polyadenylation and splicing of mRNA and the identified changes in these pathways in our study suggest mutated TIA1 affects these pathways in WDM. In addition, both WDM and TMD have common pathological changes in P70S6-mTOR signalling, SAP-JNK apoptosis and protein ubiquitination signalling pathways, which have been reported in other rimmed vacuolar myopathies.

P.3.11 Atypical phenotypes in titinopathies explained by second titin mutations and compound heterozygosity

Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe or unusual phenotypes. This study aimed to clarify the molecular cause of these variant phenotypes in proband patients of six European families. Clinical, histopathological and muscle imaging data of patients and family members was reanalyzed and muscle biopsies of the patients were studied by titin Western blotting and RT-PCR. Western blotting showed a more pronounced C-terminal titin abnormality than expected for heterozygous mutants in all six probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the two TTN alleles in biopsies of four patients, two of them with an LGMD2J phenotype. TTN was analyzed by Sanger sequencing from genomic DNA of the patients. Three patients proved to be compound heterozygotes with novel TTN frameshift mutations combined with previously reported TMD mutations. One LGMD2J patient was heterozygous for the FINmaj TMD mutation with a likely second yet undiscovered mutation. The unequal expression levels of TTN transcripts in these four probands suggested that the expression of the frameshifted allele was severely reduced, probably through nonsense-mediated decay, explaining the observed more severe phenotypes. One Portuguese patient was homozygote for a previously known Spanish TMD mutation. This TMD mutation seems to cause a more severe TMD rather than LGMD2J when homozygous. One Finnish patient had FINmaj mutation combined with a novel missense mutation in the A-band region of titin causing a new phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism of phenotype variability in muscular dystrophies.

G.O.1 Welander distal myopathy is caused by a mutated RNA binding protein

Abstract Welander distal myopathy (WDM) is a late onset, autosomal dominant distal myopathy prevalent in Finland and Sweden. First symptoms of finger extensor weakness occur after age 40, with progression to all hand muscles and ankle dorsiflexion. Rare homozygotes show also proximal muscular weakness, earlier onset and faster progression. WDM is linked to chromosome 2p13 with a maximal linked region of interest of

G.P.37 Muscle immunohistochemistry and pathology in Welander distal myopathy

Abstract Welander distal myopathy (WDM) is a dominant late-onset muscular dystrophy affecting primarily the extensor muscles of the fingers, and progressing later to involve lower leg and all hand muscles. The muscle histopathology in WDM shows variable myopathic–dystrophic changes in the affected muscles with frequent rimmed vacuoles and autophagic degenerative pathology on electron microscopic examination. We have analyzed WDM muscle biopsies using immunohistochemistry, immunofluorescence and Western blotting to further elaborate which molecular pathways are affected in WDM. We have assessed the expression of the proteins involved in the autophagosomal–lysosomal pathway (LAMP2, Cathepsin B and LC3), the ubiquitin–proteasome-mediated protein degradation pathway and VCP, the linker between these pathways. In addition, we used common markers (SMI31, p62 and TDP-43) of the rimmed vacuolar pathologies such as s-IBM. Our results suggest that there is an increase of misfolded or aggregated, partly ubiquitinated proteins as shown with increase of sequestosome (p62), TDP-43 and SMI31 both in the rimmed vacuolar regions and patchy in all atrophic fibers. This is accompanied with activation of the autophagic system as judged by some increase of LAMP2 positive regions that are too large to represent normal mature lysosomes. In contrast, the rimmed vacuoles are more or less devoid of LAMP2 positive mature lysosomes, and are instead filled with LC3-positive autophagosomes. The primary mutation in WDM causes a downstream abnormality including both incapacity of the proteasomal system to degrade all ubiquitinated proteins, and apparent decompensation of the autophagic system despite its activation.