Brochure MuscleScan. The genetic test MuscleScan specifically detects DNA regions in which the genetic changes mutations can

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1 Brochure MuscleScan MuscleScan The genetic test MuscleScan specifically detects DNA regions in which the genetic changes mutations can cause Duchenne, Emery-Dreifuss and Becker Muscular Dystrophy, Limb-Girdle Muscular Dystrophy (LGMD), Muscular Dystrophy-Dystroglycanopathy (MDDG) or Pompe disease. These are hereditary and progressive muscle diseases that have in common that one mutation in one single gene can cause all clinical features. More than 27 genes are identified that can lead to these diseases. The strength of Next Generation Sequencing (NGS) is that one single test reveals the genetic information of all genes that may be affected. All relevant DNA loci are screened simultaneously in a relatively short turn-around time, making it the best test for diagnosing these hereditary muscle diseases. Duchenne Muscle Dystrophy (DMD) usually shows a very apparent set of clinical findings which makes screening of a larger panel unnecessary. Specifically for the DMD gene, GenomeScann offers a DMD Single Gene Test, which includes also the copy number variations (CNVs) using an MLPA based test. More information can be found on our website. When testing for MuscleScan? To detect the exact location of the mutation To confirm the clinical diagnosis The specific location and mutation help to predict the disease progression Detection of the subtype can help to create the best treatment plan Which genes are includedd in MuscleScan? We determine the DNA sequence of 27 genes related to Duchenne en Becker Muscular Dystrophy (mutation in DMD), Emery- Dreifuss Muscular Dystrophy (EMD) and Limb-Girdle Dystrophy (ANO5, CAPN3, CAV3, DAG1, DES, DNAJB6, DYSF, FKRP, FKTN, IAA, ISPD, LMNA, MYOT, PLEC1, POMT1, POMT2, POMGnT1, SGCA, SGCB, SGCD, SGCG, TCAP, TNN, TNPO3, TRIM32). For more detailedd information see the table below. Muscle disease Alternative name(s) Duchenne muscular dystrophy Becker muscular dystrophy Emery-Dreifuss muscular dystrophy Limb-Girdle Muscle Dystrophyy LGMD 1A Myofibrillarr Myopathy (MFM)/ Spheroid Body Myopathy Gene name DMD DMD EMD MYOT Full name Dystrophin Dystrophin Emerin Myotilin OMIM (Disease) Brochure MuscleScan; version _EN Pagina 1 van 6

2 LGMD 1B Emery Dreifuss Muscle Dystrophy type 2 LMNA Lamin A/C LGMD 1C Rippling Muscle Disease CAV3 Caveolin LGMD 1D Myofibrillar myopathy 1 (MFM1) DES Desmin LGMD 1E DNAJB LGMD 1F TNPO3 Transportin LGMD 2A CAPN3 Calpaïne LGMD 2B LGMD 2C Miyoshi Muscle Dystrophy 1 (MMD1) Gamma- Sarcoglycanopathy DYSF Dysferlin SGCG Gamma-sarcoglycan LGMD 2D Alfa-Sarcoglycanopathy SGCA Alfa-sarcoglycan LGMD 2E Beta-Sarcoglycanopathy SGCB Beta-sarcoglycan LGMD 2F Delta-Sarcoglycanopathy SGCD Delta-sarcoglycan LGMD 2G TCAP Titin-cap (telethonin) LGMD 2H TRIM32 Tripartite motifcontaining LGMD 2I MDDGC C5 FKRP Fukutin related protein LGMD 2J TTN Titin or Connectin LGMD 2K LGMD 2L MDDGC C1 LGMD 2M MDDGC C4; LGMD 2N Miyoshi Muscle Dystrophy 3 (MMD3) Fukuyama Congenital Muscular Dystrophy; MDDGC C2 POMT1 Protein O- mannosyltransferase ANO5 Anoctamin FKTN Fukutin POMT2 Protein O- mannosyltransferase LGMD 2O MDDGC C3 POMGnT LGMD 2P MDDGC C9 DAG1 Dystrophin-associated glycoprotein MDDGC: Muscular Dystrophy-Dystroglycanopathy Type C (Amberger et al., 2011) LGMD 2Q PLEC1 Plectin Other diseases MDDGC C7 Pompe disease; Glycogen Storage Disease type II, GSD2, acid maltase deficiency ISPD Isoprenoid synthase domain-containing protein GAA Alpha-glucosidase Brochure MuscleScan; version _EN Pagina 2 van 6

3 Duchenne Muscular Dystrophy (DMD) and Becker Dystrophy are both x-linked recessive muscle diseases that are caused by mutations in the dystrophine gene. The progression of Becker Dystrophy is generally milder because in contrast to DMD, dystrophine is expressed, but in lower quantities or solely part of the protein is produced. When clinical manifestations of DMD are evident we offer the DMDx test that measures deletions and duplications in the DMD gene. Often, the clinical features are less clear-cut. Therefore, GenomeScan has developed MuscleScan, that determines the sequence of all genes in which mutations lead to a similar phenotype. The benefit of a complete panel is that all disease-causing genes are determined simultaneously within a relatively short time-frame. Limb-Girdle Muscular dystrophy (LGMD) consists of multiple subtypes (1A-F and 2A-2Q). LGMD is divided into two main groups bases on the inheritance of the disease: Limb-Girdle type 1 and 2, for respectively autosomal dominant en autosomal recessive inheritance. The majority of the patients (90%) has LGMD type 2. Every subtype is related to a mutation in one single gene. Hundreds of loci have been identified that cause muscle diseases, although no mutation hotspots have been found. Therefore, the whole coding region must be analyzed to determine the correct diagnosis. Gene name Chromosome Location Inheritance Description DMD Xp21.2-p21.1 X-linked Dystrophin; important part of the protein complex that connects the cytoskeleton of muscle fibers with the extracellular matrix through the cell membrane. EMD Xq28 X- linked Emerin localizes to the inner nuclear lamina and anchors the membrane to the cytoskeleton. LMNA 1q22 AD Codes for Lamin A en C, structural components of the nuclear lamina. MYOT 5q31.2 AD Myotilin is localized to the Z-disc of sarcomeres and binds α- Actinin. CAV3 3p25.3 AD or AR Component of the calveolar plasma-membranes that are present in most cell types. Organization and concentration of certain Caveolin-interacting molecules. CAPN3 15q15.1 AR Calcium-activating neutral protease involved in repair and regeneration of the muscle wall. DYSF 2p13.2 AR Function in calcium-dependent membrane fusions; Muscle fiber regeneration. SGCA 17q12-q21.33 AR Sarcoglycans are part of the Dystrophin-Glycoprotein complex; Muscle wall stability; Linking of Actin cytoskeleton to extracellular matrix. SGCB 4q12 AR Part of the Sarcoglycan complex (see above) SGCG 13q12 AR Part of the Sarcoglycan complex (see above) Part of the Sarcoglycan complex (see above) SGCD 5q33 AR TTN 2q31.2 AR TCAP 17q12 AR TRIM32 9q33 AR ANO5 11p14.3 AR DES 2q35 AD Titin or Connectin; in cardiac and skeletal muscles; Involved in regeneration of muscle tissue and provides mechanical resistance Solely in cardiac and skeletal muscles. Connects 2 Titin molecules, thereby augmenting the mechanical resistance. Ubiquitin ligase; Regulates DTNBP1. Causes mild LGSD. Transmembrane glycoprotein. Function currently not elucidated. Type III intermediate filament. Integrates the sarcolemma, Z-disk and nuclear membrane in sarcomeres and regulates the sarcomere composition. OMIM (Gen) Brochure MuscleScan; version _EN Pagina 3 van 6

4 TNPO3 7q32.1 AR DNAJB6 7q36.3 AD PLEC1 8q24.3 AR TCAP 17q12 AR Muscular Dystrophy-Dystroglycanopathy POMT1 9q34.13 AR POMT2 14q24.3 AR POMGnT1 1p34 AR FKTN 9q31.2 AR FKRP 19q13.1 AR Fukutin-related protein exact function not elucidated ISPD 7p21.2 AR Isoprenoid synthase domain containing protein. Necessary for efficient O-mannosylation of α-dystroglycan (DAG1) DAG1 3p21.31 AR Dystroglycan; transmembrane protein that connects the extracellular matrix with the cytoskeleton AR: Autosomal Recessive AD: Autosomal Dominant Why testing with MuscleScan? Nuclear import receptor for Serine/Arginine-rich (SR) proteins. Chaperone protein; protects proteins against irreversible aggregation during protein synthesis and in case of cellular stress. Provides mechanical resistance by crosslinking of the cytoskeleton. Solely in striated and cardiac muscle; Localization in the periphery of the Z-disk; Serves as structural anchor and has a signal function. O-mannosyltransferase enzyme in the ER; function in maintaining cell integrity and rigidity. O-mannosyltransferase enzyme in the ER; function in maintaining cell integrity and rigidity. O-mannose beta-1,2-n-acetylglucosaminyltransferase. Participates in O-mannosyl glycan synthesis Transmembrane glycosyltransferase that glycosylases a.o. α- dystroglycan in skelet muscle tissue DMD, Becker and some LGMD types can display comparable phenotypes, but the disease progression is generally dissimilar. By identifying the disease-causing mutation(s), the muscle groups that become affected over time can be predicted and the disease progression can be determined more accurately, based on patient data of the exact same genotype. - By diagnosing the exact type LGMD, the comorbidity associated with the gene defect can be monitored. Examples are cardiac- and respiratory complications, brain and eye anomalies. - Inheritance risk; the manner of inheritance and therefore the risk of passing the mutation on to offspring. - Because of psychosocial reasons patients require an accurate diagnosis that gives insight into the cause and progression of the disease. - In case of milder variants that develop during life, identifying the best type of treatment plan can differ between disease types. Because of the rapid technical innovations in NGS, the price per base becomes lower while the turnaround time becomes shorter. This opens up new possibilities to replace older and less complete tests. The publications in which pathogenic variants are linked to diseases grow rapidly. Moreover, the number of genes that are related to muscle dystrophy is increasing. Because MuscleScan is continuously updated using the latest scientific findings, it remains the most (cost-)effective manner that leads to an accurate diagnosis. Brochure MuscleScan; version _EN Pagina 4 van 6

5 Experimental workflow When patient DNA enters the GenomeScan workflow, a quality control is performed on the genomic DNA (gdna). By hybridization-capture, the coding exons including the intron-exon boundaries of the MuscleScan genes are selectively isolated. The Illumina HiSeq2500 determines the precise DNA sequence of the MuscleScan genes. This instrument is especially suited for this type of measurement because its high accuracy combined with a short sequencing time of 6 days. The GenomeScan laboratory generates all experimental findings such as the results of the quality control, sample preparation and sequencing experiments. After an extensive dataqc by GenomeScan bioinformaticians (MuscleScan Data) the bioinformaticians of the Clinical Genetics department of LUMC perform the subsequent analysis if requested (MuscleScan Plus and Total). The genetic information is compared per patient with the reference genomes, which identify the possibly disease-causing mutations per patient. All detected variations are reviewed using medical databases to determine the pathogenicity. A report letter containing the identified mutation is written by experienced laboratory specialists. Brochure MuscleScan; version _EN Pagina 5 van 6

6 Who can order this test? - Clinical geneticists of all academic hospitals - Physicians of every hospital (within the Netherlands and abroad) - Research departments of pharmaceutical or medical companies Sample requirements Patient material can be sent via the request forms on our website If you have questions regarding the sampling or amounts, please contact us via mail@genomescan.nl. Adults: Collect ng DNA in TE (10 mm Tris-Cl ph 8.0, 1 mm EDTA); conc ng/μl. Neonates: Collect 500 ng DNA in TE (10 mm Tris-Cl ph 8.0, 1 mm EDTA); conc ng/μl. Advantages of GenomeScan: - Our genetic tests are the result of extensive R&D projects and are thoroughly validated. - GenomeScan (originated from ServiceXS) has over 10 years of experiencee as sequencing service provider and therefore are experts in the genetic analysis field. - Clinical Genetics (LUMC) is an ISO15189 accredited and renowned academic diagnostic laboratory, diagnosing thousands of patients per year. - De extensive collaboration between GenomeScan and the LUMC ensuress that you have one provider for the most accurate and advanced genetic analyses. For more information contact: Info@GenomeScan.nl Tel (0) Plesmanlaan 1d, 2333 BZ Leiden Brochure MuscleScan; version _EN Pagina 6 van 6