Muscle Disorders

About muscle disorders and limb-girdle muscular dystrophy

Muscle disorders encompass a diverse range of conditions characterized by impairments in muscle function, which can manifest in various forms of weakness, stiffness, or wasting. These disorders constitute a heterogeneous group with variations in severity and age of onset. Symptoms may include muscle weakness, fatigue, cramping, and muscle stiffness. Inherited muscle disorders typically progress over time, with symptoms worsening as the condition advances.

Diagnosis of muscle disorders can be challenging due to their clinical and genetic heterogeneity. While genetic testing is often pivotal, other tests such as electromyography, muscle biopsy, blood biomarkers (CK) and imaging studies may also be employed to establish a diagnosis.

The Muscle Disorders (MD) gene panel, covering 122 genes, aids in diagnosing various muscle disorders, including muscular dystrophies with predominant limb-girdle weakness patterns, congenital muscular dystrophies, myasthenic syndromes, rigid spine syndrome, congenital myopathies, scapuloperoneal syndromes, inclusion myopathies, and metabolic myopathies, among others.

Limb-girdle muscular weakness (LGMW) is a term describing the weakness pattern encompassing a group of diseases associated with weakness and wasting of predominantly proximal muscles of the pelvic and shoulder girdles. The limb-girdle muscular dystrophies (LGMDs) encompass a heterogeneous group of disorders that vary in severity and age of onset and can be classified into two main groups depending on the inheritance pattern.  

LGMDs are clinically and genetically heterogenous and can thereby make it difficult for a physician to assign a definite diagnosis based on the clinical presentation. The Roadmap2Rare MD panel includes genes causative of overlapping clinical presentation for Limb-Girdle Muscular Dystrophy, Congenital Muscular Dystrophy, Duchenne Muscular Dystrophy, and several myopathies and myasthenic syndromes.

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Muscle Disorders

Incidence

Most LGMDs are rare, with estimated prevalence ranging from 2.4-7.3 per 100,000 (Becker) to 0.07 per 100,000 (LGMD2D, E) to 0.43 per 100,000 (LGMD2I).

Inheritance

  • Most subtypes of LGMD are autosomal recessive (LGMD2A-Q, Pompe)
  • Several rare subtypes are autosomal dominant (LGMD1A-E)
  • A few myopathies are X-linked (Becker, EGMD-X1, -X2)2

Program eligibility

The Roadmap2Rare Diagnostic Program* for Muscle Disorders (MD) testing is for individual patients with:

  • Evidence suggestive of a muscle pathology, AND
  • Muscle weakness, OR
  • Unexplained respiratory insufficiency, OR
  • Other symptom(s) suggesting muscle involvement.

*This testing program is not appropriate for carrier testing

About the test

Testing algorithm:

This panel analyzes 122 genes associated with limb girdle muscular dystrophies, other myopathies and myasthenic syndromes.  

  • NGS gene panel will be performed.  
  • Both sequencing and deletion/duplication analysis will be performed on the coding regions of all genes included (unless otherwise marked).

Genes tested

ACTA1, ACTN2, AGRN, ANO5, ATP2A1, B3GALNT2, B4GAT1, BAG3, BIN1, BVES, CACNA1S, CAPN3, CASQ1, CAV3, CCDC78, CFL2, CHAT, CHKB, CHRNA1, CHRNB1, CHRND, CHRNE, COL12A1, COL13A1, COL6A1, COL6A2, COL6A3, COLQ, CPT2, CRPPA, CRYAB, DAG1, DES, DMD, DNAJB6, DNM2, DOK7, DPAGT1, DPM3, DYSF, EMD, FHL1, FKBP14, FKRP, FKTN, FLNC, GAA, GFPT1, GMPPB, GNE, HNRNPDL, INPP5K, ITGA7, KBTBD13, KLHL40, KLHL41, KY, LAMA2, LAMP2, LARGE1, LDB3, LMNA, LMOD3, LRP4, MEGF10, MTM1, MUSK, MYBPC1, MYF6, MYH2, MYH7, MYO18B, MYOT, MYPN, NEB, ORAI1, PABPN1, PAX7, PFKM, PHKA1, PLEC, PNPLA2, POGLUT1, POMGNT1, POMGNT2, POMK, POMT1, POMT2, PYGM, RAPSN, RBCK1, RXYLT1, RYR1, SCN4A, SELENON, SGCA, SGCB, SGCD, SGCG, SLC18A3, SLC25A1, SLC5A7, SPEG, STAC3, STIM1, SYNE1, SYNE2, SYT2, TCAP, TMEM43, TNNT1, TNPO3, TOR1AIP1, TPM2, TPM3, TRAPPC11, TRIM32, TRIP4, TTN, VAMP1, VCP, VMA21

Test methods & limitations

Sequencing is performed on genomic DNA using an Agilent targeted sequence capture method to enrich for the exome. Direct sequencing of the amplified captured regions was performed using 2X150bp reads on Illumina next generation sequencing (NGS) systems. A base is considered to have sufficient coverage at 20X and an exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered at 20X or more. A list of these regions, if any, is available upon request. Alignment to the human reference genome (GRCh37) is performed and annotated variants are identified in the targeted region. Variants reviewed have a minimum coverage of 8X and an alternate allele frequency of 20% or higher. Indel and single nucleotide variants (SNVs) may be confirmed by Sanger sequence analysis before reporting at director discretion. This assay cannot detect variants in regions of the exome that are not covered, such as deep intronic, promoter and enhancer regions, areas containing large numbers of tandem repeats, and variants in mitochondrial DNA. Copy number variation (CNV) analysis detects deletions and duplications; in some instances, due to the size of the exons, sequence complexity, or other factors, not all CNVs may be analyzed or may be difficult to detect. When reported, copy number variant size is approximate. Actual breakpoint locations may lie outside of the targeted regions. CNV analysis will not detect tandem repeats, balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, and balanced insertions), methylation abnormalities, triploidy, and genomic imbalances in segmentally duplicated regions. This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director. Primary data analysis is performed using Illumina bcl2fastq converter v2.19. Secondary analysis is performed using Illumina DRAGEN Bio-IT Platform v.3.10.8. Tertiary data analysis is performed using SnpEff v5.0 and Revvity Omics' internal ODIN v.1.01 software. CNV and absence of heterozygosity are assessed using BioDiscovery’s NxClinical v6.1 software. Genes and/or exons located in pseudogene regions are not covered in this assay.

Sample requirements

DNA, isolated

Collection:

Required DNA quantity by test type*:

  • Next generation sequencing (NGS): Send >1000 ng total gDNA @ >15 ng/μL. Please ship samples in 10mM Tris. Do not use EDTA.
  • Sanger sequencing: Send >500 ng total gDNA @ >15 ng/μL (varies by the size of the gene and the variants requested).
  • Non-sanger sequencing tests: Send >500 ng total gDNA @ >15 ng/μL.

Condition: * Required DNA quality: High molecular weight DNA (>12kb). A260/A280 reading should be ≥ 1.8. A260/230 a ratio range of 1.8 to 2.2. Contact the laboratory for specific amounts if total ng cannot be met.

Shipping: Ship overnight at ambient temperature.

Special instructions:

  • Research laboratories: DNA extracted in research laboratories is not acceptable. Only under exceptional circumstances (e.g., proband not available) will DNA extracted in a research laboratory be accepted for clinical testing. Additional testing (e.g., of other family members) may be required to confirm results.
  • Laboratories outside the United States: Non-US laboratories are not subject to CLIA regulations and will be reviewed on a case-by-case basis. Please call to speak with a laboratory genetic counselor before submitting a DNA sample from any non-CLIA-certified laboratory.
  • Special notes: If extracted DNA is submitted, information regarding the method used for extraction should be sent along with the sample. 
Dried blood spots

Collection container(s): Dried blood spot card

Collection: Follow kit instructions. Briefly, allow blood to saturate the card until indicated areas are filled and blood has soaked through the card. Air dry the card at ambient temperature for at least 3 hours.

  • NBS: Please contact Revvity Omics to request the StepOne® kit.
  • Gene sequencing: Please contact Revvity Omics to request the DBS collection kit.
  • For pre-punched DBS: The required minimum is 6 punches

Condition: Follow the instructions provided with the collection set. Store the dried blood at ambient temperature for up to two days. If the specimen cannot be sent as soon as it is dry, the filter paper should be placed in a sealable plastic bag and stored in a refrigerator (≤ 8°C) or preferably in a freezer.

Shipping: Follow kit instructions. Double bag and ship overnight at ambient temperature.

Saliva

Collection container(s): Oragene™ Saliva Collection Kit or ORAcollect-Dx kit

Collection: Collect saliva on an Oragene™ Saliva Collection Kit ORAcollect-Dx kit according to the manufacturer’s instructions. 

Condition: Store at ambient temperature. Do not refrigerate or freeze.

Shipping: Ship overnight at ambient temperature.

Special instructions: Please contact Revvity Omics to request the saliva collection kit for patients who cannot provide a blood sample as whole blood is the preferred sample. Testing using Saliva swabs is currently not available for customers in India. Contact the Revvity Omics laboratory for more information.

Whole blood (EDTA)

Collection container(s): EDTA (purple top)

Collection: Infants (< 2-years): 2 to 3 mL; Children (>2-years): 3 to 5 mL; Older children and adults: Minimum 5mL. The blood tube should be inverted several times immediately after blood collection to prevent coagulation.

Condition: Store at ambient temperature. Do not refrigerate or freeze.

Shipping: Ship overnight at ambient temperature ensuring receipt within 5-days of collection.

Special instructions: Clotted or hemolyzed samples are not accepted.
 

References

1. Narayanaswami P, Weiss M, Selcen D, et al. Evidence-based guideline summary: diagnosis and treatment of limb-girdle and distal dystrophies. Neurology. 2014;83:1453-1463.

2. Murphy AP, Straub V. The classification, natural history and treatment of the limb girdle muscular dystrophies. Journal of Neuromuscular Diseases. 2015;2:S7-S19.

This testing service has not been cleared or approved by the U.S. Food and Drug Administration. Testing services may not be licensed in accordance with the laws in all countries. The availability of specific test offerings is dependent upon laboratory location. The content on this page is provided for informational purposes only, not as medical advice. It is not intended to substitute the consultation, diagnosis, and/or treatment provided by a qualified licensed physician or other medical professionals.