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Autosomal Recessive Limb Girdle Muscular Dystrophy (LGMD) 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
1347 ANO5 81406 Add to Order
CAPN3 81406
DES 81405
DYSF 81408
FKRP 81404
FKTN 81405
GAA 81406
GMPPB 81479
ISPD 81405
LIMS2 81479
PNPLA2 81479
POMGNT1 81406
POMK 81479
POMT1 81406
POMT2 81406
SGCA 81405
SGCB 81405
SGCD 81405
SGCG 81405
TCAP 81479
TOR1AIP1 81479
TRAPPC11 81479
TRIM32 81479
TTN 81479
Full Panel Price* $1890.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1347 Genes x (24) $1890.00 81404, 81405(x7), 81406(x6), 81408, 81479(x9) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

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

Many of the LGMD subtypes are ultra-rare disorders for which clinical sensitivity cannot be estimated. In a large cohort of North American LGMD patients Moore et al. (2006) made a diagnosis of dysferlinopathy in 18% of the cohort using a combined immunological and molecular approach, making LGMD2B the most common cause of LGMD in this mixed population. Childhood onset LGMD is more likely to be caused by defects in the sarcoglycan genes (Vainzof et al. 1999) and late onset LGMD often results from pathogenic variants in the ANO5 gene (Sarkozy et al. 2013).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ANO5$690.00 81479 Add to Order
CAPN3$690.00 81479
DES$690.00 81479
DYSF$690.00 81479
FKRP$690.00 81479
FKTN$690.00 81479
GAA$690.00 81479
GMPPB$690.00 81479
ISPD$690.00 81479
LIMS2$690.00 81479
PNPLA2$690.00 81479
POMGNT1$690.00 81479
POMK$690.00 81479
POMT1$690.00 81479
POMT2$690.00 81479
SGCA$690.00 81479
SGCB$690.00 81479
SGCD$690.00 81479
SGCG$690.00 81404
TCAP$690.00 81479
TOR1AIP1$690.00 81479
TRAPPC11$690.00 81479
TRIM32$690.00 81479
TTN$690.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (24) $1290.00 81404, 81479(x23) 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

Many of the genes in this panel have no or very few large deletions/duplications reported, indicating clinical sensitivity is low for this panel. However, the DYSF, GAA, and SGCG genes have a higher proportion of gross deletions/duplications reported and could be considered for aCGH testing (Human Gene Mutation Database).

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Clinical Features

Limb girdle muscular dystrophy (LGMD) is a descriptive term for a group of disorders with atrophy and weakness of proximal limb girdle muscles, typically sparing the heart and bulbar muscles. Clinical severity, age of onset, and disease progression are highly variable among the subtypes (Sáenz et al. 2005). Serum creatine kinase levels are typically elevated, and muscle biopsies demonstrate a dystrophic process. For a comprehensive review see Pegoraro and Hoffman (2012).

Genetics

Over twenty genes have been implicated in recessively inherited limb girdle muscular dystrophy: ANO5, CAPN3, DYSF, FKRP, FKTN, GMPPB, ISPD, LIMS2, PNPLA2, SGCA, SGCB, SGCD, SGCG, TCAP, TOR1AIP1, TRAPPC11, TRIM32, PLEC, POMGNT1, POMK, POMT1, POMT2, and GAA. Pathogenic variants in these genes include missense, nonsense, splicing, small deletions/insertions, and a few large deletions/duplications (Human Gene Mutation Database). The most common forms include LGMD2A, LGMD2B, and LGMD2I, caused by pathogenic variants in the CAPN3, DYSF, and FKRP genes, respectively (Moore et al. 2006; Pegoraro and Hoffman 2012). The CAV3, DES, LMNA, and TTN genes can exhibit both recessive and dominant inheritance.

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.

Of note, this test includes coverage of FKTN deep intronic variant c.647+2084G>T.

This panel does not include analysis of TTN exons 85-95. Sanger coverage of these regions is available upon request; however, very few pathogenic variants have been reported in these exons (Human Gene Mutation Database).

This test will cover over 99% of the coding exons in the indicated genes plus flanking regions.

Indications for Test

Individuals with a limb girdle pattern of muscle weakness and atrophy and a recessive inheritance pattern.

Genes

Official Gene Symbol OMIM ID
ANO5 608662
CAPN3 114240
DES 125660
DYSF 603009
FKRP 606596
FKTN 607440
GAA 606800
GMPPB 615320
ISPD 614631
LIMS2 607908
PNPLA2 609059
POMGNT1 606822
POMK 615247
POMT1 607423
POMT2 607439
SGCA 600119
SGCB 600900
SGCD 601411
SGCG 608896
TCAP 604488
TOR1AIP1 614512
TRAPPC11 614138
TRIM32 602290
TTN 188840
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Glycogen Storage Disease Type II AR 232300
Limb-Girdle Muscular Dystrophy, Type 2A AR 253600
Limb-Girdle Muscular Dystrophy, Type 2B AR 253601
Limb-Girdle Muscular Dystrophy, Type 2D AR 608099
Limb-Girdle Muscular Dystrophy, Type 2E AR 604286
Limb-Girdle Muscular Dystrophy, Type 2F AR 601287
Limb-Girdle Muscular Dystrophy, Type 2G AR 601954
Limb-Girdle Muscular Dystrophy, Type 2H AR 254110
Limb-Girdle Muscular Dystrophy, Type 2Y AR 617072
Muscular Dystrophy, Limb Girdle, Type 2C AR 253700
Muscular Dystrophy, Limb-Girdle, Type 2J AR 608807
Muscular Dystrophy, Limb-Girdle, Type 2L AR 611307
Muscular dystrophy, limb-girdle, type 2R AR 615325
Muscular dystrophy, limb-girdle, type 2S AR 615356
Muscular Dystrophy, Limb-Girdle, Type 2W AR 616827
Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7 AR 614643
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 1 AR 609308
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 12 AR 616094
Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 14 AR 615352
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 2 AR 613158
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 3 AR 613157
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 4 AR 611588
Muscular Dystrophy-Dystroglycanopathy (Limb-Girdle), Type C, 5 AR 607155
Neutral Lipid Storage Disease With Myopathy AR 610717

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Dystroglycanopathies via the POMK Gene
Dystroglycanopathy via the FKTN Gene
Dystroglycanopathy via the GMPPB Gene
Female Infertility Sequencing Panel with CNV Detection
Fukuyama Congenital Muscular Dystrophy via the FKTN Japanese Founder Mutation
Glycogen Storage Disease and Disorders of Glucose Metabolism Sequencing Panel
Glycogen Storage Disease Type II (Pompe Disease) via the GAA Gene
Glycogen Storage Disease Type II (Pompe Disease) via the GAA Gene, Exon 18 Deletion
Gnathodiaphyseal Dysplasia via the ANO5 Gene
Hypertrophic Cardiomyopathy Sequencing Panel with CNV Detection
Limb Girdle Muscular Dystrophy Type 2A via the CAPN3 Gene
Limb Girdle Muscular Dystrophy Type 2B and Miyoshi Myopathy via the DYSF Gene
Limb Girdle Muscular Dystrophy Type 2I via the FKRP Gene
Limb Girdle Muscular Dystrophy Type 2S (LGMD2S) via the TRAPPC11 Gene
Limb Girdle Muscular Dystrophy Type 2W (LGMD2W) via The LIMS2 Gene
Limb Girdle Muscular Dystrophy Type 2Y via the TOR1AIP1 Gene
Limb Girdle Muscular Dystrophy, Type 2C (LGMD2C) via the SGCG Gene
Limb Girdle Muscular Dystrophy, Type 2D (LGMD2D) via the SGCA Gene
Limb Girdle Muscular Dystrophy, Type 2E (LGMD2E) via the SGCB Gene
Limb Girdle Muscular Dystrophy, Type 2J and Tibial Muscular Dystrophy via the TTN Gene (exons 307 - 312)
Limb Girdle Muscular Dystrophy, Type 2L (LGMD2L) and Distal Miyoshi Myopathy (MMD3) via the ANO5 Gene
Limb-Girdle Muscular Dystrophy (LGMD) Sequencing Panel
Male Infertility Sequencing Panel with CNV Detection
Metabolic Myopathies, Rhabdomyolysis and Exercise Intolerance Sequencing Panel
Myofibrillar Myopathy Sequencing Panel
Myofibrillar Myopathy via the DES Gene
Neutral Lipid Storage Disease with Myopathy via the PNPLA2 Gene
Pan Cardiomyopathy Sequencing Panel with CNV Detection
Skeletal Disorders and Joint Problems Sequencing Panel with CNV Detection
Sudden Cardiac Arrest Sequencing Panel with CNV Detection
Telethoninopathy via the TCAP 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 Isoprenoid Synthase Domain Containing (ISPD) Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Human Gene Mutation Database (Bio-base).
  • Moore S.A. et al. 2006. Journal of Neuropathology and Experimental Neurology. 65: 995-1003. PubMed ID: 17021404
  • Pegoraro E., Hoffman E.P. 2012. Limb-Girdle 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: 20301582
  • Sarkozy A. et al. 2013. Human Mutation. 34: 1111-8. PubMed ID: 23606453
  • Sáenz A. et al. 2005. Brain 128: 732–42. PubMed ID: 15689361
  • Vainzof M. et al. 1999. Journal of the Neurological Sciences. 164: 44-9. PubMed ID: 10385046
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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