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Dystrophinopathy via the DMD Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1773 DMD$990.00 81408 Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

For Sanger Sequencing click here.
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Mutations detectable by sequence analysis are found in approximately one-third of DMD cases and in approximately 20% of BMD cases. In DMD cases, the vast majority of these mutations result in premature protein termination whereas missense mutations are rare (http://www.LOVD.nl/DMD).

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Deletion/Duplication testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1200 DMD$690.00 81161 Add to Order
Turnaround Time

The great majority of tests are completed within 21 days.

Clinical Sensitivity

Approximately two-thirds of the mutations in DMD patients are deletions of one or more exons in the DMD gene. The occurrence of deletions is slightly higher in BMD patients. Duplications are found in approximately 10% of DMD patients and 20% of BMD patients. In general, in-frame deletions, which preserve partial functional dystrophin protein, are correlated with a milder (BMD) clinical phenotype (Monaco et al. 1988; Aartsma-Rus et al. 2006).

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Clinical Features

The dystrophinopathies include Duchenne muscular dystrophy (DMD, OMIM 310200), Becker muscular dystrophy (BMD, OMIM 300376), and dilated cardiomyopathy, type 3B (CMD3B, OMIM 302045). 

Duchenne muscular dystrophy is the most common form of congenital muscular dystrophy in all ethnic groups. DMD presents in affected boys in early childhood with difficulty walking and climbing stairs, progressive proximal weakness, pseudohypertrophy of the calves, and greatly elevated serum creatine phosphokinase (CK) levels (Darras et al. 2011). Patients become wheelchair dependent by age 13. Dilated cardiomyopathy and congestive heart failure occur in nearly all patients by their late teens or early 20s. DMD muscle biopsies show a dystrophic process with fibrosis and fatty infiltration. Dystrophin protein content in DMD patients is less than 5% of controls when measured by Western blot or immunohistochemistry (Hoffman et al. 1988).

Becker muscular dystrophy is a relatively mild form of dystrophinopthy with later onset of proximal weakness and preservation of ambulation into the third decade of life. The minimum age for wheelchair dependency in BMD is age 16. As in DMD, cardiomyopathy is a significant cause of morbidity, however, serum CK levels are less elevated than in DMD (Zatz et al. 1991).  Dystrophin protein content in BMD patients varies from 20% of control levels to normal levels when measured by Western blot or immunohistochemistry (Hoffman et al. 1988).

Both DMD and BMD can be associated with intellectual deficits, specifically involving working memory and executive function (Darras et al. 2011). 

DMD-related cardiomyopathy in the absence of skeletal muscle disease is a less common form of dystrophinopathy which presents in affected males between 20 and 40 years of age and later in carrier females (Beggs 1997). In some mild BMD patients, cardiomyopathy can be the presenting feature (Towbin 1998).

Females who are heterozygous for a DMD causative mutation are at risk for dilated cardiomyopathy and 70% have slightly elevated CK (Schade van Westrum et al. 2011). Carriers may also present with a myopathy resembling limb-girdle muscular dystrophy (Moser and Emery 1974).

Genetics

The dystrophinopathies are inherited as X-linked recessive disorders, and approximately one-third of mothers who have a son with dystrophinopathy have no family history. Heterozygous female carriers are at increased risk for dilated cardiomyopathy. The extent of skeletal muscle involvement in carriers is dependent upon the level of skewed X-chromosome inactivation. 

The DMD gene is located at Xp21 and occupies about 1.5% of the entire X chromosome. Approximately two-thirds of the mutations in DMD patients are deletions of one or more exons in the DMD gene. The occurrence of deletions is slightly higher in BMD patients. Duplications are found in approximately 10% of DMD patients and 20% of BMD patients. In general, in-frame deletions, which preserve partial functional dystrophin protein, are correlated with a milder (BMD) clinical phenotype (Monaco et al. 1988; Aartsma-Rus  et al. 2006).

Mutations detectable by sequence analysis are found in approximately one-third of DMD cases and in approximately 20% of BMD cases. In DMD cases, the vast majority of these mutations result in premature protein termination whereas missense mutations are rare (http://www.LOVD.nl/DMD).

DMD (OMIM 300377) encodes dystrophin, a large structural protein that binds sarcoplasmic actin at its amino end and beta dystroglycan at its carboxyl end, thus bridging the cytoskeleton to the dystroglycan-associated glycoprotein complex at the sarcolemma (Barresi and Campbell 2006).

Testing Strategy

Dystrophin is coded by exons 1-79 of the DMD gene (transcript variant Dp427m; NM_004006.2) on chromosome Xp21. Testing for deletions and duplications with a high-density gene-centric array comparative genomic hybridization (aCGH) platform is Test #1200 and should be performed first.

If a mutation is not identified by aCGH, sequence analysis should be performed. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing.

Indications for Test

1. Males with a clinical diagnosis of DMD/BMD.

2. Males with symptoms of DMD/BMD (A waddling gait and difficulty climbing stairs, Gower sign, elevated CPK).

3. Male/Females with a negative DMD/BMD test by multiplex PCR, MLPA, or Southern blot.

4. Males/Females with previous DMD/BMD test result with unclear del/dup size and boundary.

5. Females at risk of being a carrier (Previous child or a family history of DMD/BMD).

Gene

Official Gene Symbol OMIM ID
DMD 300377
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Comprehensive Neuromuscular Sequencing Panel
Congenital Muscular Dystrophy Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Aartsma-Rus A, Deutekom JCT Van, Fokkema IF, Ommen G-JB Van, Dunnen JT Den. 2006. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 34: 135–144. PubMed ID: 16770791
  • Barresi R, Campbell K. 2006. Dystroglycan: from biosynthesis to pathogenesis of human disease. J. Cell Sci. 119:199-207. PubMed ID: 16410545
  • Beggs AH. 1997. Dystrophinopathy, The Expanding Phenotype Dystrophin Abnormalities in X-Linked Dilated Cardiomyopathy. Circulation 95: 2344–2347. PubMed ID: 9170393
  • Darras BT, Miller DT, Urion DK. 2011. Dystrophinopathies. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301298
  • Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterston R, Brooke M, Specht L. 1988. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N. Engl. J. Med. 318: 1363–1368. PubMed ID: 3285207
  • Leiden Open Variation Database- DMD.
  • Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM. 1988. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2: 90–95. PubMed ID: 3384440
  • Moser H, Emery AE. 1974. The manifesting carrier in Duchenne muscular dystrophy. Clin. Genet. 5: 271–284. PubMed ID: 4854942
  • Schade van Westrum SM, Hoogerwaard EM, Dekker L, Standaar TS, Bakker E, Ippel PF, Oosterwijk JC, Majoor-Krakauer DF, Essen AJ van, Leschot NJ, Wilde AAM, Haan RJ de, et al. 2011. Cardiac abnormalities in a follow-up study on carriers of Duchenne and Becker muscular dystrophy. Neurology 77: 62–66. PubMed ID: 21700587
  • Towbin JA. 1998. The role of cytoskeletal proteins in cardiomyopathies. Curr. Opin. Cell Biol. 10: 131–139. PubMed ID: 9484605
  • Zatz M, Rapaport D, Vainzof M, Passos-Bueno MR, Bortolini ER, Pavanello R de C, Peres CA. 1991. Serum creatine-kinase (CK) and pyruvate-kinase (PK) activities in Duchenne (DMD) as compared with Becker (BMD) muscular dystrophy. J. Neurol. Sci. 102: 190–196. PubMed ID: 2072118
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TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

High Resolution Deletion/Duplication testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA (gDNA) extracted from blood samples from patient (Test) and gender matched control (Reference) samples are differentially labeled with fluorescent dyes. A unique set of oligo is added to each ‘Test’ sample as spike-in. This is a quality control measure that enables us to prevent any sample mix up during the processing of the samples in the microarray lab. Following labeling both the ‘Test’ and the ‘Reference’ samples are purified, quantified and combined in equal amounts before hybridization on the microarray slides. After 22hrs of hybridization at 65°C, the slides are washed and scanned immediately using manufacturer's protocol.

The DMD array contains ‘backbone’ probes across the entire genomes, and serves to normalize data across all probes. For each patient sample the data for only the gene(s) of interest is analyzed and reported.

PreventionGenetics’ DMD array is a targeted CGH array that contains a very high density of overlapping probes providing a comprehensive coverage for both the coding and non-coding region of the entire 2.2 Mb of the DMD gene. A total of ~15,500 probes span the entire length of the DMD gene. The 79 exons of DMD totaling ~14 kb is covered by ~1500 oligo probes with an average probe spacing of 9 bp (~99% base coverage for exons). The intronic regions totaling ~2.08 Mb is covered by ~14,000 oligo probes with an average probe spacing of 150 bp (34.5% base coverage for introns). In addition, the DMD gene upstream and downstream sequences are also covered with high density probes to detect distal del/dup boundaries.

A custom aCGH based detection of deletions and duplications for DMD has increased sensitivity compared to other methods like Southern blotting, multiplex PCR, FISH and MLPA. For example, MLPA cannot detect intronic deletions and duplications which is present in 7% of the individuals with dystrophinopathies (Dent et al. 2005; Prior and Bridgeman 2005; del Gaudio et al. 2008; Hegde et al 2008). The combined DMD array and DMD full gene sequencing analysis detects ~98-99% of DMD mutations in both males and females.

Analytical Validity

PreventionGenetics’ high density DMD array enables the detection of relatively small deletions and duplications in both intronic and exonic sequences. In addition, PreventionGenetics’ DMD array design detects mosaic deletions and duplications down to 25%. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

Any intronic deletion/duplication that is not covered by our probes will not be detected by our DMD array. Only 34.5% of the intronic sequences are covered by our DMD array.

This DMD array may not detect deletions/duplications present in very low levels of mosaicism (< 25% of cells carrying deletion or duplication).

Our DMD array contains only copy number probes, and hence cannot detect Uniparental Disomy (UPD).

This DMD array cannot detect the X chromosome inactivation status, and manifesting heterozygote females will need additional X-inactivation studies.

This aCGH test will not detect balanced rearrangements such as translocations, inversions or point mutations that may lead to a clinical phenotype. This includes manifesting heterozygote females with X:Autosome translocations.

The sensitivity of this test may be reduced when DNA quality does not meet the standards established by PreventionGenetics for this assay.

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Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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