Limb Girdle Muscular Dystrophy Type 2B and Miyoshi Myopathy via DYSF Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
Nguyen et al. (Hum Mutat 26:165-175, 2005) found two DYSF mutations in 23 of 34 dysferlinopathy patients and one mutation in the remaining 11 patients. The patients were from various ethnic backgrounds and unrelated to one another. In a large cohort of North American LGMD patients Moore et al. (J Neuropathol Exp Neurol 65:995-1003) made a diagnosis of dysferlinopathy in 18% of the cohort using a combined immuno and molecular approach, making DYSF the most common cause of LGMD in this mixed population. Dysferlinopathy is also prevalent in certain ethnic groups. For example, a c.1624delG mutation underlies the high (1:1,300) prevalence for LGMD2B in Libyan Jews (Argov et al. Brain 123:1229-1237, 2000) and in Sueca, Spain a founder mutation has been found in 2% of the population (Vilchez et al. Arch Neurol 62:1256-1259, 2005). In 20 of 25 Japanese patients with a clinical diagnosis of MM, 6 different DYSF mutations were identified (Takahashi et al. Neurology 60:1799-1804, 2003).
The dysferlinopathies encompass two primary phenotypes. Limb girdle muscular dystrophy type 2B (LGMD2B; OMIM 253601) is characterized by atrophy and weakness of proximal muscles with onset in adolescence or young adulthood. Miyoshi myopathy (MM; OMIM 254130) affects distal leg muscles initially with atrophy and weakness spreading to the thighs and gluteal muscles (Miyoshi et al. Brain 109:31-54, 1986). Marked inflammatory changes are sometimes seen in muscle biopsies from MM patients. Age of onset of MM is late teens (Aoki et al. Neurology 57:271-278, 2001) and, as in LGMD2B, progression is slow. Both phenotypes exhibit massive elevations of serum CpK. Distal myopathy with anterior tibial onset (DMAT; OMIM 606768) is a third DYSF association. This phenotype has been described in a single Spanish family (Illa et al. Ann Neurol 49:130-134, 2001). It should be noted that intrafamilial variability spanning all three phenotypes has been reported (Weiler et al. Am J Hum Genet 59:872-878, 1996; Liu et al. Nat Genet 20:31-36, 1998). Approximately half of a cohort of forty dysferlinopathy patients reviewed by Nguyen et al. (Arch Neurol 64:1176-1182, 2007) had MM or LGMD2B. Another one-third of the cohort had atypical phenotypes with mixed proximal and distal weakness. Distal painful leg swelling without muscle weakness occurred in 10%. The same study reported that 25% of the patients were initially misdiagnosed as having polymyositis.
Dysferlin-related disorders are inherited in an autosomal recessive manner. Mutations are distributed throughout the gene (http://www.dmd.nl/). Nonsense, missense, small insertions and deletions and splice site mutations have been reported. Dysferlin appears to function in calcium-dependent membrane repair of skeletal muscle fibers (Bansal and Campbell Trends Cell Biol 14:206-213, 2004). Evaluation of muscle biopsies shows that most dysferlinopathy patients have complete deficiency of the protein although individuals with partial dysferlin deficiency have been reported (Piccolo et al. Ann Neurol 48:902-912, 2000). Dysferlin deficiency can also occur secondary to mutations in the genes for caveolin-3 or calpain-3. Patients with MM who have negative DYSF tests may have mutations in the ANO5 gene (Bolduc et al. Am J Hum Genet 86:213-221, 2010).
For this Next Generation Sequencing (NGS) test, 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 regions not captured or with insufficient number of sequence reads.
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.
Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.
This test provides full coverage of all coding exons of the DYSF gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Individuals with clinical symptoms consistent with LGMD, MM or DMAT. Individuals with absent staining of dysferlin in muscle or peripheral blood monocytes.
|Official Gene Symbol||OMIM ID|
|Limb-Girdle Muscular Dystrophy, Type 2B||AR||253601|
|Myopathy, Distal, With Anterior Tibial Onset||AR||606768|
- Genetic Counselor Team - firstname.lastname@example.org
- Angela Gruber, PhD - email@example.com
- Aoki, M., et.al. (2001). "Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy." Neurology 57(2): 271-8. PubMed ID: 11468312
- Argov, Z., et.al. (2000). "Muscular dystrophy due to dysferlin deficiency in Libyan Jews. Clinical and genetic features." Brain 123 ( Pt 6): 1229-37. PubMed ID: 10825360
- Bansal, D., Campbell, K. P. (2004). "Dysferlin and the plasma membrane repair in muscular dystrophy." Trends Cell Biol 14(4): 206-13. PubMed ID: 15066638
- Bolduc, V. et.al. (2010). "Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies." Am J Hum Genet 86(2): 213-221. PubMed ID: 20096397
- Illa, I., et.al. (2001). "Distal anterior compartment myopathy: a dysferlin mutation causing a new muscular dystrophy phenotype." Ann Neurol 49(1): 130-4. PubMed ID: 11198284
- Liu, J., et.al. (1998). "Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy." Nat Genet 20(1): 31-6. PubMed ID: 9731526
- Miyoshi, K., et.al. (1986). "Autosomal recessive distal muscular dystrophy as a new type of progressive muscular dystrophy. Seventeen cases in eight families including an autopsied case." Brain 109 ( Pt 1): 31-54. PubMed ID: 3942856
- Moore SA, Shilling CJ, Westra S, Wall C, Wicklund MP, Stolle C, Brown CA, Michele DE, Piccolo F, Winder TL, Stence A, Barresi R, King N, King W, Florence J, Campbell KP, Fenichel GM, Stedman HH, Kissel JT, Griggs RC, Pandya S, Mathews KD, Pestronk A, Serrano C, Darvish D, Mendell JR. 2006. Limb-girdle muscular dystrophy in the United States. J Neuropathol Exp Neurol 65: 995-1003. PubMed ID: 17021404
- Nguyen, K., et.al. (2005). "Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies." Hum Mutat 26(2): 165. PubMed ID: 16010686
- Nguyen, K., et.al. (2007). "Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes." Arch Neurol 64(8): 1176-82. PubMed ID: 17698709
- Piccolo, F., et.al. (2000). "Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb--girdle muscular dystrophies." Ann Neurol 48(6): 902-12. PubMed ID: 11117547
- Takahashi, T., et.al. (2003). "Dysferlin mutations in Japanese Miyoshi myopathy: relationship to phenotype." Neurology 60(11): 1799-804. PubMed ID: 12796534
- Vilchez, J. J., et.al. (2005). "Identification of a novel founder mutation in the DYSF gene causing clinical variability in the Spanish population." Arch Neurol 62(8): 1256-9. PubMed ID: 16087766
- Weiler, T., et.al. (1996). "Limb-girdle muscular dystrophy and Miyoshi myopathy in an aboriginal Canadian kindred map to LGMD2B and segregate with the same haplotype." Am J Hum Genet 59(4): 872-8. PubMed ID: 8808603
Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 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.
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 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.
Deletion and Duplication Testing via NGS
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.
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 ~10 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.
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.
- 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.
(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.
(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.
(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.