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Dystroglycan-Related Congenital Muscular Dystrophy Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1303 B3GALNT2 81479 Add to Order
B4GAT1 81479
DAG1 81479
DPM1 81479
DPM2 81479
DPM3 81479
FKRP 81404
FKTN 81405
GMPPB 81479
GOSR2 81479
ISPD 81405
LARGE1 81479
POMGNT1 81406
POMGNT2 81479
POMK 81479
POMT1 81406
POMT2 81406
TMEM5 81479
Full Panel Price* $1790.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1303 Genes x (18) $1790.00 81404, 81405(x2), 81406(x3), 81479(x12) 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. If you would like to order a subset of these genes contact us to discuss pricing.

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

Because of extensive phenotypic and locus heterogeneity for these disorders, clinical sensitivity is difficult to estimate. In one cohort of ninety fetuses with severe cobblestone lissencephaly and associated findings consistent with Walker-Warburg syndrome, Vuillaumier-Barrot et al. (2012), obtained a genetic diagnosis in 58 cases, or 64%. These diagnosed cases had relative frequencies as follows: POMT1 (42%), POMT2 (17%), POMGNT1 (17%), TMEM5 (9%), ISPD (9%), LARGE1 (3%) and FKRP (3%). This method should have high analytical sensitivity for all dystroglycanopathy genes with the exception of LARGE1 and ISPD, which are known to harbor gross deletions/duplications.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 B3GALNT2$690.00 81479 Add to Order
B4GAT1$690.00 81479
DAG1$690.00 81479
DPM1$690.00 81479
DPM2$690.00 81479
DPM3$690.00 81479
FKRP$690.00 81479
FKTN$690.00 81479
GMPPB$690.00 81479
GOSR2$690.00 81479
ISPD$690.00 81479
LARGE1$690.00 81479
POMGNT1$690.00 81479
POMGNT2$690.00 81479
POMK$690.00 81479
POMT1$690.00 81479
POMT2$690.00 81479
TMEM5$690.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (18) $1290.00 81479(x18) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Clinical sensitivity of deletion/duplication testing for the dystroglycanopathy genes is low. Gross copy number mutations are found at a significant frequency only in the LARGE1 and ISPD genes, and not the other dystroglycanopathy genes.

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Clinical Features

The congenital muscular dystrophies are a clinically and genetically heterogeneous group of disorders characterized by elevated serum CpK levels, muscle weakness, a dystrophic process observed in biopsied muscle, and variable associated findings such as central nervous system abnormalities, cardiac muscle involvement, skeletal effects, and developmental delay. Onset of symptoms generally occurs at birth, although some symptoms do not manifest themselves until later in life. See Sparks et al. (2012) for diagnostic strategies and a comprehensive review of the congenital muscular dystrophies.

Pathogenic variants in a growing number of proven or putative O-linked and N-linked glycosyltransferase genes and one putative lipid biosynthesis gene (ISPD, Willer et al. 2012; Roscioli et al. 2012) cause muscular dystrophies in the dystroglycanopathy spectrum. Walker-Warburg syndrome (WWS), a severe congenital muscular dystrophy with defective neuronal migration and associated structural brain and eye abnormalities, is the most severe manifestation. Other presentations include muscle-eye-brain disease (MEB), Fukuyama congenital muscular dystrophy (FCMD), and congenital or limb girdle muscular dystrophy with associated cognitive impairment, but without structural brain abnormalities (eg., LGMD2K) (Godfrey et al. 2007). Dystroglycanopathy may manifest primarily as a cardiomyopathy with minimal skeletal muscle involvement (Margeta et al. 2009).

Genetics

The dystroglycanopathies are inherited in an autosomal recessive manner. Glycosyltransferase activity is necessary for proper post translational processing of alpha dystroglycan (ADG), a protein encoded by DAG1. In the absence of these proteins, ADG remains hypoglycosylated and diverse pathologies follow (Barresi and Campbell 2006). Molecular diagnosis (and classification) of the dystroglycanopathy subtypes is complex because extensive genetic heterogeneity exists for each disorder (Godfrey et al. 2007), and because the reported phenotypes caused by the glycosyltransferase genes continue to expand (van Reeuwijk et al. 2006). Evaluation of a patient’s muscle biopsy by immunofluorescence can detect abnormal glycosylation of ADG and can, therefore, aid in a diagnostic evaluation.

See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, 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, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

This test will cover 100% of the coding exons in the indicated genes plus flanking regions.

This test includes coverage of the FKTN deep intronic Korean founder variant c.648-1243 G>T as well as c.1045-22A>G. This test will not detect the FKTN Japanese founder variant which is a 3kb retrotransposan insertion in the 3' UTR.

Indications for Test

Elevated serum CpK at birth or early in life. Hypoglycosylation of alpha dystroglycan.

Genes

Official Gene Symbol OMIM ID
B3GALNT2 610194
B4GAT1 605517
DAG1 128239
DPM1 603503
DPM2 603564
DPM3 605951
FKRP 606596
FKTN 607440
GMPPB 615320
GOSR2 604027
ISPD 614631
LARGE1 603590
POMGNT1 606822
POMGNT2 614828
POMK 615247
POMT1 607423
POMT2 607439
TMEM5 605862
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Congenital Disorder Of Glycosylation Type 1E AR 608799
Congenital Disorder Of Glycosylation Type 1O AR 612937
Congenital Disorder of Glycosylation, Type Iu AR 615042
Congenital Muscular Dystrophy-Dystroglycanopathy (With Brain And Eye Anomalies) Type A5 AR 613153
Congenital Muscular Dystrophy-Dystroglycanopathy (With Or Without Mental Retardation) Type 5B AR 606612
Fukuyama Congenital Muscular Dystrophy AR 253800
Muscle Eye Brain Disease AR 253280
Muscular Dystrophy-Dystroglycanopathy (Congenital with Brain and Eye Anomalies), Type A, 10; MDDGA10 AR 615041
Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 12 AR 615249
Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 13 AR 615287
Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 14 AR 615350
Muscular Dystrophy-Dystroglycanopathy (Congenital With Brain And Eye Anomalies), Type A, 2 AR 613150
Muscular Dystrophy-Dystroglycanopathy (Congenital With Brain And Eye Anomalies), Type A, 6 AR 613154
Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7 AR 614643
Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies, type A, 11 AR 615181
Muscular Dystrophy-Dystroglycanopathy (Congenital With Mental Retardation), Type B, 1 AR 613155
Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 14 AR 615351
Muscular Dystrophy-Dystroglycanopathy (Congenital With Mental Retardation), Type B, 2 AR 613156
Muscular Dystrophy-Dystroglycanopathy (Congenital With Mental Retardation), Type B, 3 AR 613151
Muscular Dystrophy-Dystroglycanopathy (Congenital With Mental Retardation), Type B, 6 AR 608840
Muscular Dystrophy-Dystroglycanopathy (Congenital Without Mental Retardation), Type B, 4 AR 613152
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 9 AR 613818
Walker-Warburg Congenital Muscular Dystrophy AR 236670

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Autosomal Recessive Limb Girdle Muscular Dystrophy (LGMD) Sequencing Panel
Comprehensive Neuromuscular Sequencing Panel
Congenital Disorders of Glycosylation (CDG) Sanger Sequencing Panel 2
Congenital Disorders of Glycosylation, Type Ie (CDG Ie) via the DPM1 Gene
Congenital Disorders of Glycosylation, Type Io Plus Secondary Dystroglycanopathy via the DPM3 Gene
Congenital Muscular Dystrophy Sequencing Panel
Dystroglycanopathies via the POMK Gene
Dystroglycanopathy via the B3GALNT2 Gene
Dystroglycanopathy via the DAG1 Gene
Dystroglycanopathy via the FKTN Gene
Dystroglycanopathy via the LARGE1/LARGE Gene
Dystroglycanopathy via the GMPPB Gene
Early Infantile Epileptic Encephalopathy Sequencing Panel
Early Infantile Epileptic Encephalopathy, Recessive Sequencing Panel
Limb Girdle Muscular Dystrophy Type 2I via the FKRP Gene
Limb-Girdle Muscular Dystrophy (LGMD) Sequencing Panel
Progressive Myoclonic Epilepsy via the GOSR2 Gene
Walker-Warburg Syndrome via the B3GNT1(B4GAT1) Gene
Walker-Warburg Syndrome via the POMGNT1 Gene
Walker-Warburg Syndrome via the POMT1 Gene
Walker-Warburg Syndrome via the POMT2 Gene
Walker-Warburg Syndrome via the TMEM5 Gene
Walker-Warburg Syndrome via the Glycosyltransferase-Like Domain-Containing Protein 2 (POMGNT2) Gene
Walker-Warburg Syndrome via the Isoprenoid Synthase Domain Containing (ISPD) Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Barresi R., Campbell K.P. 2006. Journal of Cell Science. 119: 199-207. PubMed ID: 16410545
  • Godfrey C. et al. 2007. Brain. 130: 2725-35. PubMed ID: 17878207
  • Margeta M. et al. 2009. Muscle & Nerve. 40: 883-9. PubMed ID: 19705481
  • Roscioli T. et al. 2012. Nature Genetics. 44: 581-5. PubMed ID: 22522421
  • Sparks S. et al. 2012. Congenital Muscular Dystrophy Overview. 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: 20301468
  • van Reeuwijk J. et al. 2006. Human Mutation. 27: 453-9. PubMed ID: 16575835
  • Vuillaumier-Barrot S. et al. 2012. American Journal of Human Genetics. 91: 1135-43. PubMed ID: 23217329
  • Willer T. et al. 2012. Nature Genetics. 44: 575-80. PubMed ID: 22522420
Order Kits
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.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

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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