Forms

Dystroglycanopathy via the DAG1 Gene

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
384 DAG1$710.00 81479 Add to Order
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 18 days.

Clinical Sensitivity
At this time there is no known clinical utility for this test.  Analytical and clinical sensitivity are unknown because there are no reported genotype-phenotype correlations for dystroglycan mutations in humans.  Findings generated from this test should not alone be considered as diagnostic.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 DAG1$690.00 81479 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 Features
Dystroglycan is the central component of the muscle dystrophin-glycoprotein complex [(DGC) Ervasti et al. Nature 345:315-319, 1990, Michele and Campbell. J Biol Chem 278:15457-15460, 2003]. Beta-dystroglycan spans the sarcolemma, binding dystrophin intracellulary and alpha-dystroglycan extracellularly. Alpha-dystroglycan is connected to the basal lamina by way of linkage to laminin (Ibraghimov-Beskrovnaya et al. Nature 355:696-702, 1992). Alpha-dystroglycan binds additional proteins with laminin G-like domains such as agrin and perlecan in muscle and neurexin in brain (Sugita et al. J Cell Biol 154:435-445, 2001). The gene DAG1 (OMIM 128239) encodes one precursor peptide that is cleaved post translationally to form the alpha and beta subunits of dystroglycan. Both subunits undergo further modification by N- and O-linked glycosylation; however, alpha-dystroglycan undergoes extensive O-linked glycosylation (Ibraghimov-Beskrovnaya et al. Hum Mol Genet 2:1651-1657, 1993). The glycosylation status of dystroglycan is critical for ligand binding as well as for pathogenesis (Michele et al. Nature 418:417-422, 2002; Muntoni et al. Curr Opin Neurol 17:205-209, 2004; Barresi and Campbell. J Cell Sci 119:199-207, 2006).
Genetics
Alpha and beta dystroglycan are encoded by the DAG1 gene located on chr 3p21. The 895 residue precursor peptide is directed to the endoplasmic reticulum via a 29 amino acid N-terminal signal peptide. Cleavage of the precursor peptide occurs at p.Ser654 to yield core alpha and beta dystroglycan proteins. A single transmembrane domain (residues 751-774) directs beta-dystroglycan to span the sarcolemma. Interaction between dystrophin and beta-dystroglycan occurs at a PPXY motif (p.Pro828_p.Tyr831) at the C-terminus of beta dystroglycan. The predicted mass of alpha-dystroglycan core protein and the corresponding protein isolated from tissue differ because of extensive species and tissue specific O-linked glycosylation of the central mucin domain between residues 312 and 485 (Barresi and Campbell, 2006). Studies in the mouse demonstrate that constitutional DAG1 mutations are lethal in the early embryonic period secondary to disruption of Reichert’s membrane (Williamson et al. Hum Mol Genet 6:831-841, 1997; Henry and Campbell Cell 95:859-870, 1998). Transgenic animals in which expression of the knockout DAG1 gene construct is limited to the brain show structural malformations like those seen in patients with congenital muscular dystrophies, as well as loss of high affinity binding to laminin (Moore et al. Nature 418:422-425).  One patient with limb girdle muscular dystrophy and severe cognitive impairment was found to be homozygous for a DAG1 mutation (p.Thr192Met, Hara et al. New Eng J Med 364: 939-946, 2011).
Testing Strategy
Testing is accomplished by amplifying the 2 coding exons of DAG1 and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard dideoxy sequencing methods and a capillary electrophoresis instrument. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
Patients with clinically or histopathologically diagnosed dystroglycanopathy. 

Gene

Official Gene Symbol OMIM ID
DAG1 128239
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Barresi, R. and Campbell, K. P. (2006). "Dystroglycan: from biosynthesis to pathogenesis of human disease." J Cell Sci 119(Pt 2): 199-207. PubMed ID: 16410545
  • Ervasti, J. M., et.al. (1990). "Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle." Nature 345(6273): 315-9. PubMed ID: 2188135
  • Hara, Y., et.al. (2011). "A dystroglycan mutation associated with limb-girdle muscular dystrophy." N Engl J Med 364(10): 939-46. PubMed ID: 21388311
  • Henry, M. D., Campbell, K. P. (1998). "A role for dystroglycan in basement membrane assembly." Cell 95(6): 859-70. PubMed ID: 9865703
  • Ibraghimov-Beskrovnaya, O., et.al. (1992). "Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix." Nature 355(6362): 696-702. PubMed ID: 1741056
  • Ibraghimov-Beskrovnaya, O., et.al. (1993). "Human dystroglycan: skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization." Hum Mol Genet 2(10): 1651-7. PubMed ID: 8268918
  • Michele, D. E., Campbell, K. P. (2003). "Dystrophin-glycoprotein complex: post-translational processing and dystroglycan function." J Biol Chem 278(18): 15457-60. PubMed ID: 12556455
  • Michele, D. E., et.al. (2002). "Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies." Nature 418(6896): 417-22. PubMed ID: 12140558
  • Moore, S. A., et.al. (2002). "Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy." Nature 418(6896): 422-5. PubMed ID: 12140559
  • Muntoni, F., et.al. (2004). "Defective glycosylation in congenital muscular dystrophies." Curr Opin Neurol 17(2): 205-9. PubMed ID: 15021250
  • Sugita, S., et.al. (2001). "A stoichiometric complex of neurexins and dystroglycan in brain." J Cell Biol 154(2): 435-45. PubMed ID: 11470830
  • Williamson, R. A., et.al. (1997). "Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1-null mice." Hum Mol Genet 6(6): 831-41. PubMed ID: 9175728
Order Kits
TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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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