New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses

Introduction: Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. Results: Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. Conclusions: Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies

New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses

Introduction: Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. Results: Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. Conclusions: Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies

Differential proteomic analysis of abnormal intramyoplasmic aggregates in desminopathy

Desminopathy is a subtype of myofibrillar myopathy caused by desmin mutations and characterized by protein aggregates accumulating in muscle fibers. The aim of this study was to assess the protein composition of these aggregates. Aggregates and intact myofiber sections were obtained from skeletal muscle biopsies of five desminopathy patients by laser microdissection and analyzed by a label-free spectral count-based proteomic approach. We identified 397 proteins with 22 showing significantly higher spectral indices in aggregates (ratio >1.8, p <0.05). Fifteen of these proteins not previously reported as specific aggregate components provide new insights regarding pathomechanisms of desminopathy. Results of proteomic analysis were supported by immunolocalization studies and parallel reaction monitoring. Three mutant desmin variants were detected directly on the protein level as components of the aggregates, suggesting their direct involvement in aggregate-formation and demonstrating for the first time that proteomic analysis can be used for direct identification of a disease-causing mutation in myofibrillar myopathy. Comparison of the proteomic results in desminopathy with our previous analysis of aggregate composition in filaminopathy, another myofibrillar myopathy subtype, allows to determine subtype-specific proteomic profile that facilitates identification of the specific disorder. Biological significance Our proteomic analysis provides essential new insights in the composition of pathological protein aggregates in skeletal muscle fibers of desminopathy patients. The results contribute to a better understanding of pathomechanisms in myofibrillar myopathies and provide the basis for hypothesis-driven studies. The detection of specific proteomic profiles in different myofibrillar myopathy subtypes indicates that proteomic analysis may become a useful tool in differential diagnosis of protein aggregate myopathies. This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations. (C) 2013 Elsevier B.V. All rights reserved

Proteomics of rimmed vacuoles define new risk allele in inclusion body myositis

OBJECTIVE: Sporadic inclusion body myositis (sIBM) pathogenesis is unknown; however, rimmed vacuoles (RVs) are a constant feature. We propose to identify proteins that accumulate within RVs. METHODS: RVs and intact myofibers were laser microdissected from skeletal muscle of 18 sIBM patients and analyzed by a sensitive mass spectrometry approach using label-free spectral count-based relative protein quantification. Whole exome sequencing was performed on 62 sIBM patients. Immunofluorescence was performed on patient and mouse skeletal muscle. RESULTS: 213 proteins were enriched by >1.5X in RVs compared to controls and included proteins previously reported to accumulate in sIBM tissue or when mutated cause myopathies with RVs. Proteins associated with protein folding and autophagy were the largest group represented. One autophagic adaptor protein not previously identified in sIBM was FYCO1. Rare missense coding FYCO1 variants were present in 11.3% of sIBM patients compared with 2.6% of controls (p=0.003). FYCO1 co-localized at RVs with autophagic proteins such as MAP1LC3 and SQSTM1 in sIBM and other RV myopathies. One FYCO1 variant protein had reduced co-localization with MAP1LC3 when expressed in mouse muscle. INTERPRETATION: This study used an unbiased proteomic approach to identify RV proteins in sIBM that included a novel protein involved in sIBM pathogenesis. FYCO1 accumulates at RVs and rare missense variants in FYCO1 are overrepresented in sIBM patients. These FYCO1 variants may impair autophagic function leading to RV formation in sIBM patient muscle. FYCO1 functionally connects autophagic and endocytic pathways supporting the hypothesis that impaired endolysosmal degradation underlies the pathogenesis of sIBM

Two novel nebulin variants in an adult patient with congenital nemaline myopathy

Congenital myopathies are clinically and genetically heterogeneous disorders, which often remain genetically undiagnosed for many years. Here we present a 40-year old patient with an almost lifelong history of a congenital myopathy of unknown cause. Muscle biopsy in childhood revealed mild myopathic features and rods. Clinical examination on presentation at the age of 40 revealed a facial weakness, atrophy and weakness of the arm muscles and distal leg muscles with mild contractures of the foot flexors and the right elbow. Subsequently, the nebulin gene was identified as a putative candidate gene by linkage analyses, but sequence analysis only revealed one heterozygous splice site mutation in intron 73 (c.10872+1G>T). Therefore, "Next Generation Sequencing" was performed, which revealed a second pathogenic variant in exon 145 (c.21622A>C). Compound-heterozygous carrier status was confirmed via sequence analysis of the index patient's parents. Whole body muscle MRI showed a muscle involvement as previously described in nebulin-associated myopathies. Based on biopsy material, genetic analyses and muscle MRI, we identified two novel, compound-heterozygous variants in the nebulin gene after a 30 year clinical history, which cause a classical childhood type of nemaline myopathy.publisher: Elsevier articletitle: Two novel nebulin variants in an adult patient with congenital nemaline myopathy journaltitle: Neuromuscular Disorders articlelink: http://dx.doi.org/10.1016/j.nmd.2015.01.013 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe

Generation of a human iPSC line (HIMRi001-A) from a patient with filaminopathy

Here we introduce the human induced pluripotent stem cell (hiPSC) line HIMRi001-A generated from cultured dermal fibroblasts of a 60-year-old male patient with a myofibrillar myopathy, carrying a heterozygous c.4984C > T [p.Q1662X] mutation in the filamin C (FLNC)-gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi001-A displays typical embryonic stem cell-like morphology, carries the c.4984C > T FLNC gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and holds the potential to differentiate in all three germ layers. We postulate that HIMRi001-A can be used for the elucidation of FLNC-associated pathomechanisms and for developing new therapeutic options

Next-generation Sequencing in der Diagnostik der genetischen Schwerhörigkeit

Hintergrund: Die genetische Heterogenität der Schwerhörigkeit erschwert die molekulargenetische Diagnostik. Neue Methoden der Hochdurchsatzsequenzierung (next-generation sequencing, NGS) erlauben es alle bisher bekannten Gene für Schwerhörigkeit in Form eines Panels von 95 Genen oder durch "Whole Exome Sequencing" simultan und umfassend zu untersuchen.Material und Methoden: Ein Kollektiv 120 schwerhöriger Personen wurde, nach Ausschluss einer Mutationen in den Genen GJB2, GJB3 und GJB6 für die Hochdurchsatzsequenzierung ausgewählt. Aus der DNA aller 120 Patienten wurden 95 Schwerhörigkeitsgene mit einem individuell entwickelten Kit angereichert und anschließend parallel sequenziert. Die hierdurch gefundenen Varianten wurden mit einer zweiten, unabhängigen Methode (klassische Sanger Sequenzierung) validiert.Ergebnisse: Durch Agilent In Solution Anreicherung und parallele Sequenzierung wurden einzelne Basenaustausche und Deletionen in bekannten Genen für erbliche Schwerhörigkeit identifiziert. Höchstwahrscheinlich pathogene Mutationen folgten in 53% einem autosomal dominanten und in 45% einem autosomal rezessiven Erbgang. In 2% der Fälle lag ein X-chromosomaler Erbgang vor. In 17% der Fälle konnten Mutationen im Zusammenhang mit syndromaler Schwerhörigkeit nachgewiesen werden. Bei 34% wurde keine pathogene Mutation in den 95 untersuchten Schwerhörigkeitsgenen identifiziert.Diskussion: Die simultane Hochdurchsatzsequenzierung von 95 bekannten Schwerhörigkeitsgenen wurde in Form eine "Hörpanels" etabliert. Die Panel-Hochdurchsatzsequenzierung eignet sich für ein kosteneffizientes Screening bei Verdacht auf genetische Schwerhörigkeit und erlaubt eine Aufklärung der genetischen Ursache in über 60% der Fälle.Anmerkung: Der Erstautor weist auf folgenden Interessenskonflikt hin: Dr. Sarah Fehr und Dr. Moritz Menzel sind Angestellte der CeGat GmbH, Tübingen; Dr. Dr. Saskia Biskup ist Gründerin und Geschäftsführerin der CeGat GmbH, Tübingen

Generation of two induced pluripotent stem cell lines (HIMRi006-A and HIMRi007-A) from Pompe patients with infantile and late disease onset

Here we present the generation of HIMRi006-A and HIMRi007-A Pompe disease (PD) patient derived human induced pluripotent stem cell (hiPSC) lines. HIMRi006-A represents an infantile onset disease (IOPD) phenotype caused by a homozygous c.307 T > G mutation in the GAA gene. HIMRi007-A is characterized by heterozygous mutations c.–32–13 T > G/c.1716C > G and is associated with an adult onset of disease symptoms (LOPD). Both lines are generated via lentiviral expression of OCT4, SOX2, KLF4, and c-MYC. The lines display a typical embryonic stem cell morphology, express pluripotency markers, retain a normal karyotype (46, XX/XY) and have the differentiation capacity in all three germ layers. Altogether, both lines provide a resource tool to the community for future in depth molecular studies of PD pathomechanism

New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses

Introduction: Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. Results: Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. Conclusions: Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies