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Bjarne Udd and colleagues show that mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 result in limb-girdle muscular dystrophy. Their studies suggest that the mutations reduce the protective anti-aggregation effects of DNAJB6, leading to protein accumulation and autophagic pathology. Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago1, 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.

Objective A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy. Methods The genetic linkage was confirmed and defined by microsatellite and single nucleotide polymorphism haplotyping. The whole linked genomic region was sequenced with targeted high‐throughput and Sanger sequencing, and coding transcripts were sequenced on the cDNA level. WDM muscle biopsies were studied by Western blotting and immunofluorescence microscopy. Splicing of TIA1 and its target genes in muscle and myoblast cultures was analyzed by reverse transcriptase polymerase chain reaction. Mutant TIA1 was characterized by cell biological studies on HeLa cells, including quantification of stress granules by high content analysis and fluorescence recovery after photobleaching (FRAP) experiments. Results The linked haplotype at 2p13 was narrowed down to <806 kb. Sequencing by multiple methods revealed only 1 segregating coding mutation, c.1362 G>A (p.E384K) in the RNA‐binding protein TIA1, a key component of stress granules. Immunofluorescence microscopy of WDM biopsies showed a focal increase of TIA1 in atrophic and vacuolated fibers. In HeLa cells, mutant TIA1 constructs caused a mild increase in stress granule abundance compared to wild type, and showed slower average fluorescence recovery in FRAP. Interpretation WDM is caused by mutated TIA1 through a dominant pathomechanism probably involving altered stress granule dynamics. Ann Neurol 2013;73:500–509

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.Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing.Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation.The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a 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 explaining phenotype variability.

Trophoblast giant cells are instrumental in promoting blood flow towards the mouse embryo by invading the uterine endometrium and remodelling the maternal vasculature. This process involves the degradation of the perivascular smooth muscle layer and the displacement of vascular endothelial cells to form trophoblast-lined blood sinuses. How this vascular remodelling is achieved at the molecular level remains largely elusive. Here, we show that two placenta-specific cathepsins, Cts7 and Cts8, are expressed in distinct but largely overlapping subsets of giant cells that are in direct contact with maternal arteries. We find that Cts8, but not Cts7, has the capacity to mediate loss of smooth muscle α-actin and to disintegrate blood vessels. Consequently, conditional ubiquitous overexpression of Cts8 leads to midgestational embryonic lethality caused by severe vascularization defects. In addition, both cathepsins determine trophoblast cell fate by inhibiting the self-renewing capacity of trophoblast stem cells when overexpressed in vitro. Similarly, transgenic overexpression of Cts7 and Cts8 affects trophoblast proliferation and differentiation by prolonging mitotic cell cycle progression and promoting giant cell differentiation, respectively. We also show that the cell cycle effect is directly caused by some proportion of CTS7 localizing to the nucleus, highlighting the emerging functional diversity of these typically lysosomal proteases in distinct intracellular compartments. Our findings provide evidence for the highly specialized functions of closely related cysteine cathepsin proteases in extra-embryonic development, and reinforce their importance for a successful outcome of pregnancy.

Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, has been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca(2+) plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca(2+) signalling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca(2+) metabolism in DM patients, including Ca(2+) channels and Ca(2+) binding proteins.We used patient muscle biopsies to analyse mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting.Most of the genes studied showed mRNA up-regulation in expression profiling. When analysed by immunohistochemistry the Ca(2+) release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca(2+) storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1.We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca(2+) metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.

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 VCP's 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.

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. 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. Myotonic dystrophy type 2 (DM2; Online Mendelian Inheritance in Man 602668) is an autosomal dominant multisystemic disease with a highly variable phenotype, characterized by adult- or late-onset proximal muscle weakness, myalgia, myotonia, cardiac conduction defects, cataracts, insulin resistance, mild cerebral involvement, and liver enzyme elevation.1Machuca-Tzili L. Brook D. Hilton-Jones D. Clinical and molecular aspects of the myotonic dystrophies: a review.Muscle Nerve. 2005; 32: 1-18Crossref PubMed Scopus (197) Google Scholar, 2Krahe R. Bachinski L. Udd B. Myotonic dystrophy type 2: clinical and genetic aspects.in: Wells R.D. Ashizawa T. Genetic Instabilities and Neurological Diseases. ed 2. Academic Press/Elsevier, Amsterdam, Boston2006: 131-150Crossref Scopus (10) Google Scholar DM2 is caused by an uninterrupted (CCTG)n expansion of between 75 and 11,000 repeats in a polymorphic (TG)n(TCTG)n(CCTG)n repeat tract in intron 1 of the CNBP gene on chromosome 3q21.3Liquori C.L. Ricker K. Moseley M.L. Jacobsen J.F. Kress W. Naylor S.L. Day J.W. Ranum L.P. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.Science. 2001; 293: 864-867Crossref PubMed Scopus (1014) Google Scholar, 4Bachinski L.L. Czernuszewicz T. Ramagli L.S. Suominen T. Shriver M.D. Udd B. Siciliano M.J. Krahe R. Premutation allele pool in myotonic dystrophy type 2.Neurology. 2009; 72: 490-497Crossref PubMed Scopus (50) Google Scholar Typical features of DM2 muscle histopathology include extreme atrophy in a subpopulation of type IIA fibers, some of them as nuclear clump fibers, and an increased amount of internal nuclei.5Vihola A. Bassez G. Meola G. Zhang S. Haapasalo H. Paetau A. Mancinelli E. Rouche A. Hogrel J. Laforet P. Maisonobe T. Pellissier J.F. Krahe R. Eymard B. Udd B. Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2.Neurology. 2003; 60: 1854-1857Crossref PubMed Scopus (139) Google Scholar DM2 is a common form of muscular dystrophy in adults, at least in some European populations.6Suominen T. Bachinski L.L. Auvinen S. Hackman P. Baggerly K.A. Angelini C. Peltonen L. Krahe R. Udd B. Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland.Eur J Hum Genet. 2011; 19: 776-782Crossref PubMed Scopus (104) Google Scholar The mutation frequency is as high as 1 in 1830 in the Finnish population,6Suominen T. Bachinski L.L. Auvinen S. Hackman P. Baggerly K.A. Angelini C. Peltonen L. Krahe R. Udd B. Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland.Eur J Hum Genet. 2011; 19: 776-782Crossref PubMed Scopus (104) Google Scholar which suggests a clinical manifestation frequency of 1 in 5000. On the basis of the late onset of the symptoms, more than one-half of mutation carriers are asymptomatic at any given time. In contrast to the more severe myotonic dystrophy type 1 (DM1), there is no congenital form of DM2, and the age of onset and disease severity is not linked to the length of the repeat expansion.7Udd B. Krahe R. The myotonic dystrophies: molecular, clinical, and therapeutic challenges.Lancet Neurol. 2012; 11: 891-905Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar DM2 pathogenesis has been shown to result from an RNA gain-of-function pathomechanism that involves sequestration of trans-acting nuclear proteins, such as muscleblind-like 1, which co-localizes with mutant RNA repeats in ribonuclear foci.8Sallinen R. Vihola A. Bachinski L.L. Huoponen K. Haapasalo H. Hackman P. Zhang S. Sirito M. Kalimo H. Meola G. Horelli-Kuitunen N. Wessman M. Krahe R. Udd B. New methods for molecular diagnosis and demonstration of the (CCTG)n mutation in myotonic dystrophy type 2 (DM2).Neuromuscul Disord. 2004; 14: 274-283Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar This leads to missplicing of a range of effector genes, including INSR,9Savkur R.S. Philips A.V. Cooper T.A. Dalton J.C. Moseley M.L. Ranum L.P. Day J.W. Insulin receptor splicing alteration in myotonic dystrophy type 2.Am J Hum Genet. 2004; 74: 1309-1313Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar CLCN1,10Mankodi A. Takahashi M.P. Jiang H. Beck C.L. Bowers W.J. Moxley R.T. Cannon S.C. Thornton C.A. Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy.Mol Cell. 2002; 10: 35-44Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar BIN1,11Fugier C. Klein A.F. Hammer C. Vassilopoulos S. Ivarsson Y. Toussaint A. Tosch V. Vignaud A. Ferry A. Messaddeq N. Kokunai Y. Tsuburaya R. de la Grange P. Dembele D. Francois V. Preciquot G. Boulade-Ladame C. Hummel M.C. Lopez de Munain A. Sergeant N. Laquerriere A. Thibault C. Deryckere F. Auboeuf D. Garcia L. Zummermann P. Udd B. Schoser B. Takahashi M.P. Nishino I. Bassez G. Laporte J. Furling D. Charlet-Berquerand N. Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.Nat Med. 2011; 17: 720-725Crossref PubMed Scopus (243) Google Scholar CACNA1S,12Tang Z.Z. Yarotskyy V. Wei L. Sobczak K. Nakamori M. Eichinger K. Moxley R.T. Dirksen R.T. Thornton C.A. Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of Ca(V)1.1 calcium channel.Hum Mol Genet. 2012; 21: 1312-1324Crossref PubMed Scopus (126) Google Scholar and other genes,13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar, 14Nakamori M. Sobczak K. Puwanant A. Welle S. Eichinger K. Pandya S. Dekdebrun J. Heatwole C.R. McDermott M.P. Chen T. Cline M. Tawil R. Osborne R.J. Wheeler T.M. Swanson M.S. Moxley 3rd, R.T. Thornton C.A. Splicing biomarkers of disease severity in myotonic dystrophy.Ann Neurol. 2013; 74: 862-872Crossref PubMed Scopus (172) Google Scholar which likely contribute to the phenotypic features in patients with DM2. However, other mechanisms, such as decreased CCHC-type zinc finger, nucleic acid binding protein (CNBP) expression may also have a role in the DM2 pathology.15Raheem O. Olufemi S.E. Bachinski L.L. Vihola A. Sirito M. Holmlund-Hampf J. Haapasalo H. Li Y.P. Udd B. Krahe R. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.Am J Pathol. 2010; 177: 3025-3036Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Patients who take statins have a dose-related and time-dependent increased risk of adverse muscle reactions and rhabdomyolysis.16Rallidis L.S. Fountoulaki K. Anastasiou-Nana M. Managing the underestimated risk of statin-associated myopathy.Int J Cardiol. 2011; 159: 169-176Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar There is also a clear increase in the frequency of adverse events during combination therapy or if there is an underlying subclinical myopathy.17Laaksonen R. On the mechanisms of statin-induced myopathy.Clin Pharmacol Ther. 2006; 79: 529-531Crossref PubMed Scopus (26) Google Scholar Patients with DM2 frequently have hyperlipidemia and often require statin treatment.18Udd B. Meola G. Krahe R. Thornton C. Ranum L. Bassez G. Kress W. Schoser B. Moxley R. 140th ENMC International Workshop: myotonic dystrophy DM2/PROMM and other myotonic dystrophies with guidelines on management.Neuromuscul Disord. 2006; 16: 403-413Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 19Heatwole C. Johnson N. Goldberg B. Martens W. Moxley 3rd, R. Laboratory abnormalities in patients with myotonic dystrophy type 2.Arch Neurol. 2011; 68: 1180-1184Crossref PubMed Scopus (27) Google Scholar Among the patients diagnosed with DM2 in Finland, a larger than average proportion had statin-induced adverse muscle reactions, including the occasional rhabdomyolysis. Certain polymorphisms in SLCO1B120Vladutiu G.D. Isackson P.J. SLCO1B1 variants and statin-induced myopathy.N Engl J Med. 2009; 360 (letter to the editor): 304Crossref PubMed Scopus (21) Google Scholar have been associated with an increased risk of statin-induced myopathy. Global expression profiling of skeletal muscles of patients without myopathy on statin treatment has shown changes in the calcium regulatory and the membrane repair machinery.21Draeger A. Sanchez-Freire V. Monastyrskaya K. Hoppeler H. Mueller M. Breil F. Mohaupt M.G. Babiychuk E.B. Statin therapy and the expression of genes that regulate calcium homeostasis and membrane repair in skeletal muscle.Am J Pathol. 2010; 177: 291-299Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In addition, changes in the cholesterol metabolism pathways in muscle cell lines treated with statins have been reported.22Morikawa S. Murakami T. Yamazaki H. Izumi A. Saito Y. Hamakubo T. Kodama T. Analysis of the global RNA expression profiles of skeletal muscle cells treated with statins.J Atheroscler Thromb. 2005; 12: 121-131Crossref PubMed Scopus (58) Google Scholar Our aim was to identify molecular factors that increase the susceptibility of patients with DM2 to statin-adverse muscle reactions by comparing the expression profiles in three different muscle biopsy sets. Expression profiles of DM2 muscle biopsies were compared with previously published DM2 muscle expression profiles13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar and profiles of patients with hyperlipidemia with no muscle disorders, taking simvastatin.23Laaksonen R. Katajamaa M. Päivä H. Sysi-Aho M. Saarinen L. Junni P. Lütjohann D. Smet J. Van Coster R. Seppänen-Laakso T. Lehtimäki T. Soini J. Oresic M. A systems biology strategy reveals biological pathways and plasma biomarker candidates for potentially toxic statin-induced changes in muscle.PLoS One. 2006; 1: e97Crossref PubMed Scopus (195) Google Scholar Our hypothesis was that similar molecular pathways would be affected in patients with DM2 and by statin treatment. In this combined analysis we found 21 genes with shared dysregulated expression. In addition, the prevalence and relevance of the risk-associated polymorphism in SLCO1B1 was analyzed in a large Finnish DM2 cohort. All patients with DM1 and with DM2 were from Finland and were diagnosed by DNA mutation testing.4Bachinski L.L. Czernuszewicz T. Ramagli L.S. Suominen T. Shriver M.D. Udd B. Siciliano M.J. Krahe R. Premutation allele pool in myotonic dystrophy type 2.Neurology. 2009; 72: 490-497Crossref PubMed Scopus (50) Google Scholar Altogether nine different DM2 patient samples were studied (clinical information is summarized in Table 1). The main clinical symptoms of the patients with DM2 consisted of muscle pain and stiffness, especially after exercise, and proximal muscle weakness that was more pronounced in the lower limbs. Only two patients had clinically detectable myotonia. Half of the patients had hyperlipidemia, but none had diabetes mellitus. Whole genome expression array analysis was performed on six DM2 and four control biopsies from the vastus lateralis muscle with the use of the Illumina expression array platform (Illumina, Inc., San Diego, CA) (Table 1). For the comparison, we included previously published expression studies on DM2 patient biopsies13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar and simvastatin-treated hyperlipidemic persons.23Laaksonen R. Katajamaa M. Päivä H. Sysi-Aho M. Saarinen L. Junni P. Lütjohann D. Smet J. Van Coster R. Seppänen-Laakso T. Lehtimäki T. Soini J. Oresic M. A systems biology strategy reveals biological pathways and plasma biomarker candidates for potentially toxic statin-induced changes in muscle.PLoS One. 2006; 1: e97Crossref PubMed Scopus (195) Google Scholar Biopsies of the patients with DM2 A and B (Table 1) were used both in the Illumina DM2 array study and in the previously reported Affymetrix DM2 study.13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar The study was approved by the institutional review board of Tampere University Hospital, and all patients gave written informed consent.Table 1Patient and Control Biopsies Used in Illumina Gene Expression Analysis, RT-PCR, and Western Blot AnalysisNameSex/age at biopsy (years)MethodMuscle symptomsClinical findingsHyperlipidemiaDiabetesMyotonic dystrophy type 2 Patient AM/49EAExercise-induced myalgiaNormal muscle strength, calf hypertrophyYesNo Patient BM/38EAExercise-induced myalgia and weaknessClinical myotonia, mild proximal lower limb atrophyYesNo Patient C (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.1)F/34EAMuscle stiffness, clumsinessNormal muscle strength, myotonia on EMGNoNo Patient D (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.2)F/41EADifficulties in climbing stairs, muscle stiffnessProximal lower limb weakness, clinical myotoniaNoNo Patient E (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.3)M/55EAExercise-induced myalgia, muscle stiffnessProximal weakness and atrophy, calf hypertrophy, myotonia on EMGNoNo Patient F (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.4)M/37EAMuscle stiffnessMild proximal weakness, myotonia on EMGYesInsulin resistance DM2-A (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.5)M/44PCRExercise-induced myalgia and weakness, muscle stiffnessMild proximal weaknessNANo DM2-B (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.4)M/37PCRSee patient F DM2-CF/63PCRMild bent spine syndromeAxial and proximal weaknessYesNo DM2-DM/51PCRDifficulties in climbing stairs, muscle stiffnessProximal weakness, myotonia on EMGNANo D1 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.3)M/55WBSee patient E D2 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.1)F/34WBSee patient C D3 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.2)F/41WBSee patient D D4 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.5)M/44WBSee DM2-AControls Ctrl AM/52EA Ctrl BM/45EA Ctrl CM/50EA Ctrl DM/54EA C-1M/79PCR C-2M/80PCR C-3F/UPCR C1U/>65WB C2U/>65WBMyotonic dystrophy type 1 DM1-AM/34PCR DM1-BF/42PCR DM1-CM/50PCR DM1-DF/47PCRF, female; M, male; Ctrl, control; DM1, myotonic dystrophy type 1, DM2, myotonic dystrophy type 2; EA, expression array; EMG, electromyography; NA, not available; U, unknown; WB, Western blot analysis.∗ Overlapping biopsies indicating the same patient's biopsy was present in different experiments. Open table in a new tab F, female; M, male; Ctrl, control; DM1, myotonic dystrophy type 1, DM2, myotonic dystrophy type 2; EA, expression array; EMG, electromyography; NA, not available; U, unknown; WB, Western blot analysis. Muscle biopsies were homogenized with ultra-turrax (IKA turrax, S8N-5 G), and total RNA was extracted with Trizol (15596-018; Invitrogen, Carlsbad, CA) and purified with the RNeasy kit (74106; Qiagen, Valencia, CA). The total RNA was treated with DNase (79254; Qiagen) according to the manufacturer's recommendations. One hundred nanograms of total RNA was amplified and biotinylated with the use of the Illumina RNA Total Prep Amplification kit (AMIL1791; Ambion, Austin, TX) for 14 hours. The Illumina gene expression array analysis was performed at the Microarray Center at Turku University by using 1.50 μg of sample RNA with Sentrix Human-6 Expression BeadChips version 2 (BD-25-113; Illumina, Inc.) at 58°C overnight (17 hours) according to Illumina whole genome gene expression with IntelliHyb Seal-protocol, revision B. The hybridization was detected with 1 μg/mL cyanine 3–streptavidine (PA43001; GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The expression arrays were scanned with Illumina BeadArray Reader and analyzed with Bead Studio version 3. The expression array raw intensity signals were analyzed with Inforsense KDE version 2.0.4 (Inforsense, London, UK) by using quantile normalization. This software was used for single-gene analyses, including fold-change calculations and filtering of the probes. Statistical significance was calculated with an unpaired t-test. Probes that did not reach statistical significance (P ≤ 0.05) were removed from the data. The microarray data are available from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE45331). Pathway changes were identified with the Database for Annotation, Visualization and Integrated Discovery ontology database (http://david.abcc.ncifcrf.gov, last accessed January 24, 2011) with KEGG (Kyoto Encyclopedia of Genes and Genomes) downstream annotations. Significant pathway enrichment in the DM2 expression data were calculated with EASE score (modified Fisher exact t-test) with multiple testing correction (Benjamini–Hochberg). The pathways were ranked with the EASE score t-test (P ≤ 0.05). Genomic DNA was extracted from peripheral blood leukocytes by using the QIAamp DNA Blood Mini-kit. The SLCO1B1 single nucleotide polymorphism (SNP) rs4149056 was genotyped with TaqMan genotyping assays (Applied Biosystems, Foster City, CA). Random duplicates were used as controls. cDNA was generated with 1 μg of total RNA from myotube cultures or from muscle tissue by using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. In vitro transcription was performed with random hexamers and oligo-(dT) priming according to the manufacturer's instructions (SuperScript III First-strand cDNA Synthesis Kit; Invitrogen). The PCR products were amplified with DreamTaq master mix (EP0702; Fermentas, Burlington, ON, Canada) and were separated on an agarose gel. PCR products were identified by sequencing of representative DNA bands. Primer sequences are given in Table 2.Table 2A List of Primers Used in RT-PCR AnalysisOligonucleotideSequenceNEDD4 006-1L5′-CTCCTCCTCCTCCACAGTTG-3′NEDD4 006-1R5′-CGGTGCTGCTGAGGATGA-3′NEDD4Fwd: 5′-TTGCAGCAACAACAAGAACC-3′Rev: 5′-GCAAGTCCAGGCGATTAAAA-3′Nedd4Fwd: 5′-CACAGTGGAAAAGGCCAAGC-3′Rev: 5′-ACCTGGTGGCAATCCAGATG-3′GAPDHFwd: 5′-TTAGCACCCCTGGCCAAGG-3′Rev: 5′-CTTACTCCTTGGAGGCCATG-3′Fwd, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEDD4, neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase; Rev, reverse. Open table in a new tab Fwd, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEDD4, neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase; Rev, reverse. Quantification of the cDNA was performed with TaqMan-based quantitative real-time PCR by using NEDD4 (Hs00406454_m1), Gapdh (Mm99999915_g1), and GAPDH (4333764F) primers and probes (Life Technologies, Carlsbad, CA). TaqMan master mix (10 mL; Applied Biosystems), 0.5 μL of 1:10 diluted cDNA, and 2 μL of primer and probe sets were used in a 20-μL total reaction volume. Amplification and detection were performed with the ABI 7500 system (Applied Biosystems). The PCR thermal conditions were 50°C for 2 minutes, 95°C for 10 seconds, and 60°C for 1 minute. Each sample was performed in triplicate and normalized to GAPDH by using standard curves for each gene on the same plate. Muscle biopsies were prepared for SDS-PAGE by homogenization with 19 volumes of sample buffer that contained 4 mol/L urea and 4% SDS at 100°C for 5 minutes. Samples were resolved with 12% SDS-PAGE gels with 4% stacking gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Antibodies used in immunoblotting, immunofluorescence (IF), and immunohistochemistry (IHC) are rabbit monoclonal anti–phosphatase and tensin homologue (PTEN; IF and IHC dilution 1:100; 6H2.1; Millipore, Billerica, MA), rabbit polyclonal anti-ubiquitin (dilution 1:150; Z0458; Dako UK Ltd., Ely, UK), mouse anti-dystrophin (dilution 1:50; Dy4/6D3; Novocastra, Newcastle, UK), polyclonal rabbit anti-NEDD4 (dilution 1:1000; NBP1-03462; Novus Biologicals, Inc., Littleton, CO), mouse anti-GAPDH (dilution 1:20,000; ab8245; Abcam, Cambridge, MA), and rabbit monoclonal anti-MBNL1 (dilution 1:1000; ab108519; Abcam). Primary antibodies were detected with secondary horseradish peroxidase–conjugated antibodies (dilution 1:100; DAKO P260; DakoCytomation, Glostrup, Denmark) and enhanced chemiluminescence with the Immun-Star kit (Bio-Rad Laboratories). Secondary horseradish peroxidase–conjugated antibodies were diluted at 1:1000 in Western blot analysis. Cell culture of biopsies was performed as described previously.15Raheem O. Olufemi S.E. Bachinski L.L. Vihola A. Sirito M. Holmlund-Hampf J. Haapasalo H. Li Y.P. Udd B. Krahe R. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.Am J Pathol. 2010; 177: 3025-3036Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Immunoprecipitation (IP) assays of PTEN were done according to the manufacturer's instructions (Pierce cross-linking IP kit; Thermo Fisher Scientific, Inc., Waltham, MA). PTEN was captured with the anti-PTEN antibody (dilution 1:100; ab32199; Abcam) and identified by Western blot analysis as described above. DM2 muscle biopsies (n = 6) and control muscle biopsies (n = 2) were snap frozen in liquid nitrogen-cooled isopentane for 6 μm cryosections on SuperFrost. IHC staining was performed with the NovoLink Min Polymer detection system (reference RE7290-K; Leica Microsystems, Milton Keynes, UK) by using the aforementioned antibodies. IF was performed as previously described15Raheem O. Olufemi S.E. Bachinski L.L. Vihola A. Sirito M. Holmlund-Hampf J. Haapasalo H. Li Y.P. Udd B. Krahe R. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.Am J Pathol. 2010; 177: 3025-3036Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar by using Alexa Fluor 680 donkey anti-mouse (A10038; Invitrogen) and Alexa Fluor 405 goat anti-rabbit (A31556; Invitrogen) secondary antibodies (dilution 1:1000). Fluorescence was detected with an Axioplan Imager 2 microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) with a high-resolution, cooled camera. AxioVision version 4.6 (Carl Zeiss) was used for image acquisition. Simvastatin (S6196; Sigma-Aldrich, St. Louis, MO) was converted into the active acid according to the manufacturer's instructions. C2C12 myoblasts were grown in Dulbecco's modified Eagle's medium (Gibco 41965; Invitrogen) with high glucose (4.5 g/L) and supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin. Myoblasts were seeded in T25 flasks for 2 days until fully confluent and differentiated in Dulbecco's modified Eagle's medium with high glucose that was supplemented with penicillin, streptomycin, and 2% horse serum. After 1 week simvastatin-containing media were added to the myotubes (no treatment, ethanol vehicle, 1 μmol/L, and 10 μmol/L) in quadruplicate. The cells were harvested after 6 hours, and RNA was extracted with an RNeasy kit (Qiagen). cDNA was then produced, and PCR products were amplified with mouse primers. Cells were also harvested after 2 days of simvastatin treatment, and total protein was extracted for Western blot analysis. Band intensities were quantified with ImageJ version 1.46f (NIH, Bethesda, MD). Significance of changes between treatment groups was calculated with a two-tailed equal variance t-test. Plasmids for shRNA expression that target all protein-coding isoforms of MBNL1 and a scramble control were purchased from GeneCopoeia (Rockville, MD). Two combined shRNAs were used that contained the target sequence n7 (5′-CAATTGCAACCGAGGAGAA-3′) or n8 (5′-AGATCAAGGCTGCCCAATA-3′) in the HSH011081-7-mU6 vector. HEK293T cells were plated on 6-well plates at 150,000 cells per well and transfected with 2 μg of combined MBNL1 shRNA plasmids or a scramble control (CSHC TR001-mU6) by using FuGene 6 (Promega, Madison, WI) and OptiMem (Life Technologies) according to the manufacturer's instructions. Cells were harvested 72 hours after transfection for Western blot analysis and RNA extraction. Eight patients referred to the Neuromuscular Research Center (Tampere University and University Hospital, Finland) because of simvastatin-induced muscle symptoms (muscle pain/weakness and elevated levels of creatine kinase, including one case of rhabdomyolysis) proved to have DM2 on genetic examination. In the same 2004 to 2007 period, a total of 89 patients were diagnosed with DM2. Twelve of these patients were diagnosed in family studies, 77 were primary referrals of undetermined muscle disease, and among them were the eight patients with simvastatin-adverse reactions. Thus, 10% of the primary referred patients who proved to have DM2 had simvastatin-induced symptoms as the main cause of referral. During the 2008 to 2009 period, 19 further patients were referred for neuromuscular evaluation because of statin-induced muscle symptoms, of which 4 patients (21%) had a subsequent genetic diagnosis of DM2. Genotyping of the known myopathy-associated SNP rs4149056 in SLCO1B120Vladutiu G.D. Isackson P.J. SLCO1B1 variants and statin-induced myopathy.N Engl J Med. 2009; 360 (

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. 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.

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. To identify these components we used gene expression profiling from muscle biopsies samples of TMD patients, healthy controls and from phenotypically overlapping Welander distal myopathy (WDM) patients. The profiling results were confirmed through RT-PCR and protein level analysis and we identified an activation of the unfolded protein response (UPR). UPR was then confirmed through elevation of marker genes HSPA5 (BIP) and XBP1 and the presence of ER-stress specific XBP1 splicing events. However, UPR activation appears to be insufficient, leading to build-up of ubiquitinated proteins which in turn cause activation of the autophagic system. Massive accumulation of LC3b positive autophagosomes within the rimmed vacuolated regions of degenerated muscle fibers suggests that this apparently compensatory mechanism is not capable of restoring equilibrium since these fibers degenerate further and disappear in the end stage of the pathology cascade.

Hyperlipidaemia is frequently associated with insulin resistance in Myotonic dystrophy type 2 (DM2) patients, but the treatment of these patients is problematic, due to an increased risk of statin-induced myopathy. In our neuromuscular centre, we have noted a 10-fold increase in susceptibly of DM2 patients to statin induced myopathy. We have examined the frequency of the previously published SlCO1B1 risk allele in DM2 patient biopsies and can exclude it as a major cause of increased statin adverse reaction in DM2 patients in Finland. In this study, we compared the global gene expression profiles of muscle biopsies from DM2 patients versus control muscles and expression changes associated with individuals who are on statin medication, with the aim of distinguishing shared affected pathways in a common pathomechanism. The microarray expression profile leads were further analysed in multiple DM2 biopsies by RT-PCR, with the aim of identifying aberrantly spliced genes among the abnormally expressed genes. We identified a unique set of dysregulated genes. NEDD4, an ubiquitin ligase, was one of the dysregulated genes in DM2 patient muscles and in individuals with statin-induced changes. Our work demonstrates that NEDD4 is abnormally spliced in DM2, which leads to an aberrant isoform of NEDD4 protein being expressed in DM2 patient muscles. PTEN, a known NEDD4 target is increased at the protein level and PTEN accumulates in damaged highly atrophic DM2 muscle fibres. PTEN is linked to lipid metabolism via phosphoinositide signalling and Statins have been shown to cause a general increase in expression of PTEN in normal individuals. We are in the process of over-expressing in cell culture wild-type NEDD4 and DM2 specific splice isoforms and comparing their ability to regulate the turnover of PTEN through ubiquination. Our results suggest that the NEDD4-PTEN ubiquitination pathway becomes dysregulated in DM2 patient’s muscles.

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 <800 kb. Initially all genes were sequenced, later the whole linked genomic region was sequenced with targeted high throughput- and Sanger sequencing, and all known transcripts from the region were sequenced on the cDNA level. Finally a presumably causative gene defect, was revealed in a linked gene. The mutation was not seen in 202 normal Finnish control samples, but was present in all 56 analysed WDM samples previously known to carry the linked haplotype. The gene is coding for an RNA binding protein, that has not been reported mutated in any neuromuscular or other disorders. Immunohistochemical analysis showed predominantly nuclear localization of the protein with some cytoplasmic labelling. Western blotting showed a weaker band in muscle tissue from a homozygote WDM patient indicating either a change of protein tertiary structure with decreased antibody affinity or a decreased amount of total protein. The known functions of the gene are related to cell responses in stress suggest that understanding its malfunctions in muscle disease may have implications for many other degenerative disorders.

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.

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. 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.

Introduction: The subcellular cascade of molecular events leading from the causative titin mutations to the preterm death of muscle cells in Tibial muscular dystrophy (TMD) and Limb-Girdle muscular dystrophy type 2J (LGMD2J) is unknown. In order to identify downstream involvement of molecules in the pathogenetic mechanism we explored the Welander muscular dystrophy (WDM) and TMD biopsies genetic profile by whole genome profiling. Method: Expression profiling of 11 TMD, 7 WDM patient biopsies and 6 healthy muscle control biopsies have been completed along with bioformatic analysis of the a specific pathway/phenotypes trends associated with them. The raw data of both distal myopathies was processed and analysed using Chipster and Ingenuity Pathway Analysis. Results: Sets of unique biochemical and genetic changes characteristic for TMD and WDM, such as ATM signalling, EIF2 signalling and clathrin mediated Endocytosis signalling in TMD and melatonin signalling, GNRH signalling and purine metabolism in WDM were uncovered. We also identified shared changes that result in the similarity seen in these distal myopathies, such as mitochondrial dysfunction, protein Ubiquitination and oxidative phosphorylation changes. We report heterogenous dysregulation in two pathways in both disorders (SAP-Jnk apoptosis) and in TMD (unfolded protein response linked ER stress) by semi-quantitive RT-PCR and western blotting. Conclusion: TMD has previously been reported to have NF-KB linked apoptotic changes in dystrophic tissue. We have identified a range of significantly upregulated apoptotic pathway molecules in TMD and we show that SAP-Jnk apoptotic signalling is common to the pathology of TMD and WDM. FINMaj mutations in TMD also causes induction of ER stress markers (BIP) and lysosomal components (LAMP2).