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• W2147598379 abstract "Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation. Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation. Limb-girdle muscular dystrophies are a group of heterogeneous disorders typically showing an autosomal recessive mode of inheritance, progressive muscle weakness, and high serum creatine kinase levels.1Bonnemann CG McNally EM Kunkel LM Beyond dystrophin: current progress in the muscular dystrophies.Curr Opin Pediatr. 1996; 8: 569-582Crossref PubMed Scopus (69) Google Scholar Two types of muscle disease, a distal myopathy (Miyoshi Myopathy, MM) and a form of limb girdle muscular dystrophy (LGMD2B) have both been shown to be caused by mutations of the dysferlin gene.2Liu J Aoki M Illa I Wu C Fardeau M Angelini C Serrano C Urtizberea JA Hentati F Hamida MB Bohlega S Culper EJ Amato AA Bossie K Oeltjen J Bejaoui K McKenna-Yasek D Hosler BA Schurr E Arahata K de Jong PJ Brown Jr, RH Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nat Genet. 1998; 20: 31-36Crossref PubMed Scopus (744) Google Scholar, 3Han R Campbell KP Dysferlin and muscle membrane repair.Curr Opin Cell Biol. 2007; 19: 409-416Crossref PubMed Scopus (195) Google Scholar In both LGMD2B and MM, dysferlin gene mutations result in partial or complete loss of dysferlin protein in muscle as measured by immunoassays, although the reduction in dysferlin levels does not strictly correlate with clinical severity.4Saito A Higuchi I Nakagawa M Saito M Hirata K Suehara M Yoshida Y Takahashi T Aoki M Osame M Miyoshi myopathy patients with novel 5′ splicing donor site mutations showed different dysferlin immunostaining at the sarcolemma.Acta Neuropathol (Berl). 2002; 104: 615-620PubMed Google Scholar Both the proximal (LGMD2B) and distal (MM) phenotypes can be caused by identical mutations in the same family, suggesting the role of genetic modifiers and/or environmental influence on disease expression.5Weiler T Bashir R Anderson LV Davison K Moss JA Britton S Nylen E Keers S Vafiadaki E Greenberg CR Bushby CR Wrogemann K Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene.Hum Mol Genet. 1999; 8: 871-877Crossref PubMed Scopus (161) Google Scholar The dysferlin gene is localized to 2p13 with expression in various tissues ranging from kidney to monocytes, with highest levels in skeletal and cardiac muscle.6Han R Bansal D Miyake K Muniz VP Weiss RM McNeil PL Campbell KP Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.J Clin Invest. 2007; 117: 1805-1813Crossref PubMed Scopus (136) Google Scholar Dysferlin is localized predominantly to the muscle surface membrane, and is also associated with cytoplasmic vesicles.7Anderson LV Davison K Moss JA Young C Cullen MJ Walsh J Johnson MA Bashir R Britton S Keers S Argov Z Mahjneh I Fougerousse F Beckmann JS Bushby KM Dysferlin is a plasma membrane protein and is expressed early in human development.Hum Mol Genet. 1999; 8: 855-861Crossref PubMed Scopus (241) Google Scholar The dysferlin protein was originally named based on its similarity to the Caenorhabditis elegans protein FER-1. FER-1 is responsible for mediating fusion of intracellular vesicles with the spermatid plasma membrane.8Achanzar WE Ward S A nematode gene required for sperm vesicle fusion.J Cell Sci. 1997; 110: 1073-1081PubMed Google Scholar Sequence homology between FER-1 and dysferlin includes tandem C2 domains and a C terminal transmembrane domain. The similarity in primary structure of the two proteins led to the suggestion that dysferlin may also play a role in membrane fusion events in skeletal muscle cells.9Bashir R Britton S Strachan T Keers S Vafiadaki E Lako M Richard I Marchand S Bourg N Argov Z Sadeh M Mahjneh I Marconi G Passos-Bueno MR Moreira Ede S Zatz M Beckmann JS Bushby K A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42Crossref PubMed Scopus (552) Google Scholar C2 domains are known to bind calcium, phospholipids or proteins to trigger signaling events and membrane trafficking, and this has led to speculation that dysferlin is important for membrane repair in skeletal muscle.2Liu J Aoki M Illa I Wu C Fardeau M Angelini C Serrano C Urtizberea JA Hentati F Hamida MB Bohlega S Culper EJ Amato AA Bossie K Oeltjen J Bejaoui K McKenna-Yasek D Hosler BA Schurr E Arahata K de Jong PJ Brown Jr, RH Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nat Genet. 1998; 20: 31-36Crossref PubMed Scopus (744) Google Scholar, 10Rizo J Sudhof TC C2-domains, structure and function of a universal Ca2+-binding domain.J Biol Chem. 1998; 273: 15879-15882Crossref PubMed Scopus (705) Google Scholar This hypothesis is supported by the finding that dysferlin deficient patient muscle shows numerous structural membrane defects when analyzed by electron microscopy, including tears in the plasma membrane and an accumulation of subsarcolemmal vesicles and vacuoles.11Selcen D Stilling G Engel AG The earliest pathologic alterations in dysferlinopathy.Neurology. 2001; 56: 1472-1481Crossref PubMed Scopus (132) Google Scholar Also, laser-induced membrane damage in dysferlin-deficient myofibers has highlighted reduced membrane resealing capability compared to normal muscle myofibers.12Bansal D Miyake K Vogel SS Groh S Chen CC Williamson R McNeil PL Campbell KP Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature. 2003; 423: 168-172Crossref PubMed Scopus (757) Google Scholar These findings are consistent with a plasma membrane repair defect in dysferlin-deficient myofibers. The current understanding of molecular and cellular defects in LGMD2B/MM does not explain a number of the enigmatic features of the presentation and progression of patients with dysferlin deficiency in muscle. First, dysferlin-deficient patients are typically quite healthy until their late teens, and in our patient cohort some patients showed impressive athletic skill at young age, before disease onset. Second, disease onset is typically in late teens or early twenties, and is often associated with a subacute onset of weakness, with marked muscle inflammation on muscle biopsy.13Confalonieri P Oliva L Andreetta F Lorenzoni R Dassi P Mariani E Morandi L Mora M Cornelio F Mantegazza R Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study.J Neuroimmunol. 2003; 142: 130-136Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 14Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (175) Google Scholar The onset and inflammation often leads to the misdiagnosis of polymyositis.14Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (175) Google Scholar, 15Nguyen K Bassez G Krahn M Bernard R Laforet P Labelle V Urtizberea JA Figarella-Branger D Romero N Attarian S Leturcq F Pouget J Levy N Eymard B Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes.Arch Neurol. 2007; 64: 1176-1182Crossref PubMed Scopus (203) Google Scholar Finally, the relatively wide inter- and intrafamilial variation in clinical phenotype, subacute onset, and marked inflammation suggest that environmental factors, other genetic modifiers, or both may play a stronger role in LGMD2B than in the other dystrophies. The aggressive inflammation often observed in dysferlin-deficient muscle distinguishes it from other Limb-girdle dystrophies. Systematic studies of the inflammatory cell content showed a predominance of macrophages, and CD4+ cells, however CD8+ cytotoxic T cells are also abundant.11Selcen D Stilling G Engel AG The earliest pathologic alterations in dysferlinopathy.Neurology. 2001; 56: 1472-1481Crossref PubMed Scopus (132) Google Scholar, 13Confalonieri P Oliva L Andreetta F Lorenzoni R Dassi P Mariani E Morandi L Mora M Cornelio F Mantegazza R Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study.J Neuroimmunol. 2003; 142: 130-136Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 14Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (175) Google Scholar Normal monocytes contain dysferlin, and LGMD2B/MM patients lack dysferlin in their monocytes.16Ho M Gallardo E McKenna-Yasek D De Luna N Illa I Brown Jr, RH A novel, blood-based diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi myopathy.Ann Neurol. 2002; 51: 129-133Crossref PubMed Scopus (91) Google Scholar, 17De Luna N Freixas A Gallano P Caselles L Rojas-Garcia R Paradas C Nogales G Dominguez-Perles R Gonzalez-Quereda L Vilchez JJ Marquez C Bautista J Guerrero A Salazar JA Pou A Illa I Gallardo E Dysferlin expression in monocytes: a source of mRNA for mutation analysis.Neuromuscul Disord. 2007; 17: 69-76Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar We have recently shown that dysferlin-deficient monocytes display abnormal signaling and phagocytotic activity that could contribute to excessive inflammation in patient muscle.18Nagaraju K Rawat R Veszelovszky E Thapliyal R Kesari A Sparks S Raben N Plotz P Hoffman EP Dysferlin deficiency enhances monocyte phagocytosis: a model for the inflammatory onset of limb-girdle muscular dystrophy 2B.Am J Pathol. 2008; 172: 774-785Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Further, human LGMD2B and mouse (SJL) dysferlin-deficient muscle showed macrophage and dendritic cell activation markers, including HLA-DR, HLA-ABC, and CD86 (human), and MOMA-2, CD11c, and ICAM-1 (mice) suggesting that mild myofiber damage in dysferlin-deficient muscle may result in an exaggerated monocyte/dendritic cell response secondary to dysferlin protein loss. In the present study, we sought to identify the novel networks induced as a direct consequence of dysferlin deficiency. Key to our approach is the filtering out of molecular changes resulting from non-specific muscle pathology (degeneration, regeneration, fibrosis, and inflammation). To accomplish this, we compared a series of dysferlin-deficient, mutation-positive LGMD2B/Miyoshi patient muscle biopsy profiles to a muscular dystrophy of similar age of onset and clinical severity, LGMD2I, involving partial loss-of-function of FKRP (fukutin-related protein). We also included muscle biopsies from normal volunteers, Becker muscular dystrophy, and amyotrophic lateral sclerosis as additional controls. This approach led to the identification of both vesicle trafficking and inflammatory changes that were specific to or strongly exaggerated in dysferlin-deficient muscle. The patient population in the study was taken from a molecular diagnostic referral population. Frozen muscle biopsies from patients with a tentative diagnosis of muscular dystrophy were sent to the Hoffman laboratory at Children's National Medical Center, Washington DC. All samples were subjected to a standardized set of biochemical and histological assays. Tests included hematoxylin and eosin histological stains, dystrophin immunostaining, α-sarcoglycan immunostaining, merosin (laminin alpha2) immunostaining, dystrophin immunoblotting and dysferlin immunoblotting, as previously described.19Kesari A Pirra LN Bremadesam L McIntyre O Gordon E Dubrovsky AL Viswanathan V Hoffman EP Integrated DNA, cDNA, and protein studies in Becker muscular dystrophy show high exception to the reading frame rule.Hum Mutat. 2008; 29: 728-737Crossref PubMed Scopus (71) Google Scholar Patients were recruited and samples were analyzed under protocol 2405, which has been reviewed and approved by the Office for the Protection of Human Subjects at Children's National Medical Center. Muscle biopsies from four groups of subjects were studied for mutation and expression profiling studies: dysferlin deficient, FKRP (LGMD2I), Becker muscular dystrophy, normal volunteers (exercise studies), and amyotrophic lateral sclerosis (ALS). For dysferlin-deficient biopsies used in this study, twenty five patient biopsies showed complete or greatly reduced dysferlin signal by duplicate immunoblots (Table 1), and these patients were then used for subsequent dysferlin DNA mutation studies, mRNA profiling, and protein characterizations. For LGMD2I subjects, approximately 1000 frozen muscle biopsies from the diagnostic muscle tissue bank were selected, genomic DNA prepared from muscle cryosections, and mutation screening for the FKRP gene was done (see Materials and Methods). For Becker muscular dystrophy, patient biopsies showed dystrophin of abnormal size and quantity by duplicate immunoblots, and showed dystrophin gene deletion mutations. For ALS, patients were diagnosed by El Escorial criteria. Normal controls were from normal volunteers enrolled in exercise studies, and different biopsies used for mRNA profiling and protein validation studies.Table 1Biochemical and Molecular Features with Mutations in Dysferlin Deficient (LGMD2B/MM) and FKRP (LGMD2I) PatientsPNSexAge at biopsy (yr)Dysferlin deficiencyNucleotide changeOutcome of mutationNovel mutationMicroarrayLGMD2B/Miyoshi SubjectsD1F19Completec.610C>Tp.Arg203XNoYesc.4192_4193insCp.Cys1398SerfsX11YesD2FNACompletec.3113G>Ap.Arg1038GlnNoYesc.1749_1750insTp.Glu584XYesD3F31Completec.2779delGp.Ala927LeufsX21YesYesc.4253G>Ap.Gly1418AspYesD4M27Completec.3992G>Tp.Arg1331LeuNoYesc.3972C>TESED5F42Completec.2902A>Tp.Met968LeuYesYesc.3181delCp.Gln1061ArgfsX59YesD6F41Completec.539dupCp.Ala181GlyfsX29YesYesc.1402C>Tp.Arg468CysYesD7M41Completec.2779delGp.Ala927LeufsX21YesYesc.857T>Ap.Val286GluYesD8M20Partialc.6124C>TpArg2042CysNoYesNDD9F12Completec.1178_1180+dupsplice MutationYesYesNDD10M16Completec.1834C>Tp.Gln612XNoYes180 bp intronic deletionD11M16Partialc.5194G>Cp.Glu1732GlnYesNoc.3181delCp.Gln1061ArgfsX59YesNoD12F34Partialc.2997G>Tp.Trp999CysNoNoNDD13M54Partialc.5430–2A>Gsplice MutationYesNoNDD14M43Completec.4943A>Gp.Tyr1648CysYesNoc.4943A>Gp.Tyr1648CysYesNoD15F18Completec.3113G>Ap.Arg1038GlnNoNoNDD16M21Partialc.6124C>TpArg2042CysNoNoNDD17F26Completec.2643 + 1G>Asplice MutationNoNoc.4167 + 1G>Csplice MutationNoNoD18M37Completec.2641A>Cp.Thr881ProNoNoNDD19M22Completec.5835_5838delp.Gln1946TrpfsX18NoNoc.5835_5838delp.Gln1946TrpfsX18NoNoLGMD2I/FKRP SubjectsF1F25Normalc.826C>Ap.Leu276IleNoYesc.586G>Cp.Gly196ArgNoF2F16Normalc.826C>Ap.Leu276IleNoYesc.826C>Ap.Leu276IleNoF3M12Normalc.826C>Ap.Leu276IleNoYesNDF4M40Normalc.427C>Ap.Arg143SerNoYesNDF5F22Normalc.427C>Ap.Arg143SerNoYesNDF6M10Normalc.427C>Ap.Arg143SerNoYesc.427C>Ap.Arg143SerNoF7M6Normalc.826C>Ap.Leu276IleNoYesc.826C>Ap.Leu276IleNoF8F31Normalc.826C>Ap.Leu276IleNoYesc.826C>Ap.Leu276IleNoF9MNANormalc.826C>Ap.Leu276IleNoYesNA, Not available, ND, Not detected, PN, Patient Number. Open table in a new tab NA, Not available, ND, Not detected, PN, Patient Number. For dysferlin-deficient patients, twenty five patients showing complete or greatly reduced dysferlin by duplicate immunoblots were selected, and genomic DNA was isolated from 10 mg of flash-frozen muscle biopsy using Genomic DNA Purification Kit (Gentra Systems Minneapolis, MA). 10 ng of the genomic DNA was used as a template for amplification of each of the 55 exons of the dysferlin gene using dysferlin specific intronic primers. Primers were designed using the PRIMER 3 software and amplification was performed using ABI Ampli Taq Gold reagents (Foster City, CA). PCR products were screened for base changes by using denaturing high-pressure liquid chromatography on a Transgenomic Wave DNA Fragment Analysis System (San Jose, CA).20Kaler SG Devaney JM Pettit EL Kirshman R Marino MA Novel method for molecular detection of the two common hereditary hemochromatosis mutations.Genet Test. 2000; 4: 125-129Crossref PubMed Scopus (19) Google Scholar Melting temperature profiles were determined by sequence analysis of PCR products by using WaveMaker 4.0 software (Transgenomics, Inc., San Jose, CA). For regions containing potential heteroduplexes by denaturing high-pressure liquid chromatography, direct, automated sequencing was performed by using cycle sequencing reactions (Big Dye TM Terminator v3.1; Applied Biosystems, Foster City, CA). An ABI 3130-Genetic Analyzer (Applied Biosystems) was used for sequencing. Data were analyzed with Sequencher TM 4.1.4 (Gene Codes Corporation, Ann Arbor, MI). For LGMD2I patients, genomic DNA was extracted as described above from approximately 1000 muscle biopsies. Each DNA sample were first screened for the common mutation [c.826C>A (p.Leu276Ile)]21Brockington M Yuva Y Prandini P Brown SC Torelli S Benson MA Herrmann R Anderson LV Bashir R Burgunder JM Fallet S Romero N Fardeau M Straub V Storey G Pollitt C Richard I Sewry CA Bushby K Voit T Blake DJ Muntoni F Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.Hum Mol Genet. 2001; 10: 2851-2859Crossref PubMed Google Scholar using a TaqMan allele discrimination assay. Those showing heterozygosity for p.Leu276Ile were then subjected to DNA sequencing. Mutation numbering is based on DYSF or FKRP cDNA (with sequence position 1 being the A in the first ATG codon). A “c.” and “p.” prefix, respectively, denotes to cDNA and protein sequences. The reference sequences used were derived from GenBank ID NM_003494.2 (Dysferlin), or GenBank ID NM_024301.2 (FKRP). Mutations were detected by sequencing both strands and were confirmed by resequencing of newly amplified PCR products. All mutations have been deposited in the Leiden Muscular Dystrophy database (Center for Human and Clinical Genetics, Leiden University Medical Center; ). RNA was extracted from ten dysferlin deficient, nine fukutin-related protein (FKRP) mutation positive samples, eleven normal volunteers, and seven ALS muscle biopsy samples using the Trizol (Invitrogen Carsland, CA) method followed by further purification on RNeasy columns. Isolated RNA from each individual sample was used to prepare biotinylated cRNA target according to manufacture protocols (Affymetrix, CA). Twenty micrograms of biotinylated cRNAs from samples were fragmented and hybridized to 37 individual Affymetrix GeneChip HG U133 Plus2 (Affymetrix, Santa Clara, CA) for 16 hours. The arrays were washed and stained on the Affymetrix Fluidics station 400, using instructions and reagents provided by Affymetrix. This involves removal of non-hybridized material, and then incubation with phycoerythrin-streptavidin to detect bound cRNA. The signal intensity was amplified by second staining with biotin labeled anti-streptavidin antibody followed by phycoerythrin- streptavidin staining. Fluorescent images were captured using Hewlett-Packard G2500A gene Array Scanner. All of the arrays used in the study passed the quality control set by Tumor analysis group 2004.22Expression profiling–best practices for data generation and interpretation in clinical trials.Nat Rev Genet. 2004; 5: 229-237Crossref PubMed Scopus (203) Google Scholar Initial data analysis of Affymetrix microarrays was done using Genechip GX software (version 7.3.1), with standard operating procedures and quality controls as described previously (Tumor analysis group 2004).22Expression profiling–best practices for data generation and interpretation in clinical trials.Nat Rev Genet. 2004; 5: 229-237Crossref PubMed Scopus (203) Google Scholar Normalized signals (expression values) were calculated using three probe set algorithms, DNA-Chip Analyzer 1.7 model based expression (dChip/mismatch model),23Li C Wong WH Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection.Proc Natl Acad Sci USA. 2001; 98: 31-36Crossref PubMed Scopus (2701) Google Scholar Microarray Suite 5 (MAS5) (Affymetrix) probe set algorithms, and Probe Logarithmic Intensity Error, SDK Version 2 (PLIER) (Affymetrix). We have previously reported that PLIER and dCHIP difference model probe set algorithms provide a good signal/noise ratio in human muscle expression profiling projects,24Seo J Bakay M Chen YW Hilmer S Shneiderman B Hoffman EP Interactively optimizing signal-to-noise ratios in expression profiling: project-specific algorithm selection and detection p-value weighting in Affymetrix microarrays.Bioinformatics. 2004; 20: 2534-2544Crossref PubMed Scopus (108) Google Scholar, 25Seo J Hoffman EP Probe set algorithms: is there a rational best bet?.BMC Bioinformatics. 2006; 7: 395Crossref PubMed Scopus (76) Google Scholar and we also added MAS5 probe set algorithm as this remains the most commonly used. CEL processed image files were analyzed separately for MAS5, dCHIP, and PLIER probe set algorithms. For the study of molecular markers of inflammatory cell subsets and activation states, and for analysis of transcripts previously reported as differentially regulated, a candidate gene approach was used (See Supplemental Table S1 at ). For non-candidate (global) analysis approaches, we used concordance for three probe set algorithms and a relatively stringent P value threshold (P < 0.005) for each of the three probe set algorithms. Muscle biopsies selected for protein validation studies included a subset of those used for microarray studies (dysferlin-deficient, FKRP [LGMD2I]), as well as additional biopsies from Becker muscular dystrophy, Juvenile Dermatomyositis patients and for normal controls that showed no histological or biochemical abnormality. Serial 6 μm-thick frozen muscle sections were cut with an IEC Minotome cryostat, mounted to Superfrost Plus Slides (Fisher Scientific Pittsburgh, PA). Sections were then blocked for 30 minutes in 10% horse serum and 1× PBS and incubated with respective antibodies for 2 hours at room temperature. Mouse monoclonal antibody against versican (US Biological Swampscott, MA) and tenascin-C (Sigma St. Louis, MO) at the 1:1500 dilutions and myoferlin at 1:20 dilutions were used.26Jaiswal JK Marlow G Summerill G Mahjneh I Mueller S Hill M Miyake K Haase H Anderson LV Richard I Kiuru-Enari S McNeil PL Simon SM Bashir R Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.Traffic. 2007; 8: 77-88Crossref PubMed Scopus (48) Google Scholar Antibodies used included a rabbit polyclonal antibody against Rab27A (IBL, Japan) at the concentration of 2 μg/ml along, a mouse monoclonal antibody against laminin α 2 (Chemicon, CA) at 1:500, and a mouse monoclonal antibody against HLA-DR (DakoCytomation, CA) at 1:50. Washes were done with 10% horse serum and 1× PBS and sections were then incubated with respective secondary antibody, Cy3 and Cy2 conjugated goat anti-mouse antibody and Cy3 conjugated donkey anti-rabbit (Jackson Immunoresearch Inc. Westgrove, PA) at a 1:500 dilution. Isotype-matched mouse and rabbit Igs were used instead of primary antibodies as negative controls. Confocal images were captured on the inverted Olympus microscope equipped with a ×40 (DApo 40UV/340: NA 1.30) or ×60 (Splan Apo; NA 1.4) objective lens, Bio-rad confocal hardware (Bio-rad MRC 1024 ES, Bio-rad Hercules, CA), and a 15mW air-cooled Cr/Ar laser for excitation. Cy3 and Cy2 were detected using standard protocols for Cy3 and Cy2 antibody respectively. Biopsy cryosections were solubilized with 100 μl JSB buffer (0.1 M Tris pH 8.0, 10% SDS, 50 mmol/L DTT, 10 mmol/L EDTA, pH 8.0, and 0.02% bromophenol blue). The lysate was boiled for 5 minutes and then centrifuged at 13,000 × g at 4°C for 5 minutes. Ten microliters of each supernatant was run on a ready-cast 4% to 15% Tris-HCl polyacrylamide gel (Biorad) with protein markers (Kaleidoscope Precision Plus protein standard, Biorad, CA). Samples were then transferred onto Hybond C Extra membrane (Amersham, Little Chalfont, UK), blots blocked in 5% milk in Tris- buffered saline with 0.5% Tween 20, and then incubated with primary antibodies (below), and horseradish peroxidase-conjugated secondary antibodies. Slp2a-SHD antibody was affinity-purified rabbit IgG, used at 2 μg/ml concentration for immunoblot.27Kuroda TS Fukuda M Ariga H Mikoshiba K The Slp homology domain of synaptotagmin-like proteins 1–4 and Slac2 functions as a novel Rab27A binding domain.J Biol Chem. 2002; 277: 9212-9218Crossref PubMed Scopus (175) Google Scholar Rab27A antibody was also affinity purified rabbit IgG and used at 5 μg/ml concentration for immunoblot. Myoferlin immunoblotting was done using a mouse monoclonal supernatant26Jaiswal JK Marlow G Summerill G Mahjneh I Mueller S Hill M Miyake K Haase H Anderson LV Richard I Kiuru-Enari S McNeil PL Simon SM Bashir R Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.Traffic. 2007; 8: 77-88Crossref PubMed Scopus (48) Google Scholar at dilution of 1:50. Dysferlin immunoblotting was done with a mouse monoclonal at a dilution of 1:200 (NCL-Hamlet-2, Novocastra). After incubation three washes with TBST followed by incubation in respective horseradish peroxidase-conjugated secondary antibodies (Biorad). Detection of protein bands was performed using ECL detection kit (Amersham) and Hyperfilm ECL (Amersham) X-ray film. We characterized a series of LGMD patient muscle biopsies at both the biochemical and genetic level, with the goal of defining molecular networks specific to dysferlin-deficient muscle (LGMD2B or MM). Twenty-five subjects showing complete or partial dysferlin deficiency by immunoblot analyses of muscle biopsies were studied for dysferlin gene mutations (Table 1). Genomic DNA was isolated from each biopsy and tested for mutations of all 55 exons of the dysferlin gene using denaturing high-pressure liquid chromatography and sequencing. Nineteen of the 25 subjects studied tested positive for gene mutations (Table 1). Out of these 19 mutation-positive patients, two patients were homozygous, nine (47%) were compound heterozygotes, and in eight patients we were able to detect only one mutation. In all, 26 distinct sequence variants were identified and 13 (50%) were novel to this report. For LGMD2I patients, approximately 1000 muscle biopsies were first tested for the common mutation in FKRP gene [c.826C>A (p.Leu276Ile)] using a Taqman all" @default.
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• W2147598379 date "2008-11-01" @default.
• W2147598379 modified "2023-10-13" @default.
• W2147598379 title "Dysferlin Deficiency Shows Compensatory Induction of Rab27A/Slp2a That May Contribute to Inflammatory Onset" @default.
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