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Nemaline Myopathy Sequencing Panel
- Summary and Pricing
- Clinical Features and Genetics
- Citations
- Methods
- Ordering/Specimens
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TEST METHODS
- NextGen Sequencing using PG-Select Capture Probes
- Deletion/Duplication Testing via Array Comparative Genomic Hybridization
NGS Sequencing
| Test Code | Test Copy Genes | CPT Code Copy CPT Codes | |
|---|---|---|---|
| 1367 | ACTA1 | 81479 | Add |
| CFL2 | 81479 | ||
| KBTBD13 | 81479 | ||
| KLHL40 | 81479 | ||
| KLHL41 | 81479 | ||
| LMOD3 | 81479 | ||
| NEB | 81408 | ||
| TNNT1 | 81479 | ||
| TPM2 | 81479 | ||
| TPM3 | 81479 | ||
| Full Panel Price* | $1620.00 | ||
| Test Code | Test Copy Genes | Total Price | CPT Codes Copy CPT Codes | |
|---|---|---|---|---|
| 1367 | Genes x (10) |
$1620.00 | 81408, 81479(x9) | Add |
Pricing Comment
We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.
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 20 days.
Clinical Sensitivity
Nebulin gene pathogenic variants are most common cause of Nemaline Myopathy, accounting for up to 50% of cases (Ryan et al. 2001; North and Ryan 2002). ACTA1 pathogenic variants account for 15%-25% of all individuals with NM (Laing et al. 2009). Eight other genes (TPM3, TNNT1, TPM2, CFL2, LMOD3, KBTBD13, KLHL40, KLHL41) are involved with NM, however, the fraction of cases attributed to each of them is small. Deletion of exon 55 of NEB occurs with a carrier frequency of ~1% among people of Ashkenazi Jewish ancestry (Anderson et al. 2004).
Deletion/Duplication Testing via aCGH
| Test Code | Test Copy Genes | Individual Gene Price | CPT Code Copy CPT Codes | |
|---|---|---|---|---|
| 600 | ACTA1 | $990.00 | 81479 | Add |
| CFL2 | $990.00 | 81479 | ||
| KBTBD13 | $990.00 | 81479 | ||
| KLHL40 | $990.00 | 81479 | ||
| KLHL41 | $990.00 | 81479 | ||
| NEB | $990.00 | 81479 | ||
| TNNT1 | $990.00 | 81479 | ||
| TPM2 | $990.00 | 81479 | ||
| TPM3 | $990.00 | 81479 | ||
| Full Panel Price* | $1290.00 | |||
| Test Code | Test Copy Genes | Total Price | CPT Codes Copy CPT Codes | |
|---|---|---|---|---|
| 600 | Genes x (9) |
$1290.00 | 81479(x9) | Add |
Pricing Comment
|
# of Genes Ordered |
Total Price |
|
1 |
$990 |
|
2-5 |
$1190 |
|
6-10 |
$1290 |
|
11-100 |
$1490 |
|
Over 100 |
Call for quote |
Turnaround Time
The great majority of tests are completed within 20 days.
Clinical Sensitivity
Clinical sensitivity for deletion/duplication testing is expected to be low. One exception is the Ashkenazi Jewish nebulin exon 55 deletion.
Clinical Features
Nemaline myopathy (NM) is a genetically and clinically heterogeneous disorder characterized by muscle weakness, hypotonia and the presence of nemaline bodies in skeletal muscle fibers. Muscle weakness is typically observed in affected neonates or infants, although later onset cases are reported (Ryan et al. 2001). The most severely affected muscle groups are proximal limb, facial, bulbar, and respiratory muscles. Deep tendon reflexes are absent or depressed. Histologically, NM is characterized by type 1 fiber predominance and the presence of rod-like structures called nemaline bodies upon Gomori trichrome staining of skeletal muscle (Ryan et al. 2003). Six clinical types of NM have been delineated based on age of onset, severity and distribution of weakness, and respiratory function (Ryan et al. 2001; North and Ryan. 2015). Overlap among the six clinical groups is significant and adults are sometimes diagnosed only after another family member has presented with typical signs. Nebulin gene pathogenic variants most often cause typical neonatal onset disease, although NEB pathogenic variants have been found in every clinical form of NM (Lehtokari et al. 2006). Troponin T1 associated NM is a lethal disorder described only in the Old Order Amish community of Pennsylvania (Johnston et al. 2000). Nemaline myopathy-8 due to pathogenic variants in the KLHL40 gene represents a severe form of this disease with features of fetal akinesia or hyopkinesia and early lethality (Ravenscroft et al. 2013).
Genetics
Pathogenic variants in a growing number of genes have been shown to cause nemaline myopathy. Most forms of nemaline myopathy (NEB, CFL2, KLHL40, KLHL41, LMOD3 and TNNT1) are inherited as autosomal recessive diseases, although ACTA1 or TPM3-related nemaline myopathy can be inherited as a dominant or recessive condition. TPM2 and KBTBD13-related nemaline myopathy are inherited as autosomal dominant conditions (Nowak et al 2015). Pathogenic variants in NEB and ACTA1 are the only relatively common causes accounting for up to 50% and 15-25%, respectively, of all studied cases (Ryan et al. 2001; North and Ryan 2015). The majority of reported NEB pathogenic variants (Lehtokari et al. 2014) are splice site variants (34%), frameshift (32%), and nonsense (23%). Reported pathogenic missense variants appear to be less common (~7%). The only common NEB pathogenic variant is an exon 55 deletion found at a carrier frequency of about 1% among people of Ashkenazi Jewish ancestry (Anderson et. al. 2004). ACTA1 missense variants are a major type of reported dominant pathogenic variants and are commonly associated with sporadic (de novo) cases. Recessive ACTA1-related myopathy is very rare, usually due to null ACTA1 pathogenic variants, in the compound heterozygous state with pathogenic missense variants (Laing et al. 2009; http://www.LOVD.nl/ACTA1). See individual gene test descriptions for information on molecular biology of gene products.
Testing Strategy
For this NextGen test, the full coding regions plus ~10 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.
Exons 82-105 of the NEB gene are organized in three highly homologous blocks of 8 exons each making this region difficult to analyze by NextGen Sequencing only. We employ a novel longe-range 2-step PCR with Sanger sequencing method to analyze this region of the NEB gene which allows correct exon and zygosity assignment.
Indications for Test
Individuals with clinical symptoms consistent with nemaline myopathy and a muscle biopsy with nemaline bodies.
Genes
| Official Gene Symbol | OMIM ID |
|---|---|
| ACTA1 | 102610 |
| CFL2 | 601443 |
| KBTBD13 | 613727 |
| KLHL40 | 615340 |
| KLHL41 | 607701 |
| LMOD3 | 616112 |
| NEB | 161650 |
| TNNT1 | 191041 |
| TPM2 | 190990 |
| TPM3 | 191030 |
| Inheritance | Abbreviation |
|---|---|
| Autosomal Dominant | AD |
| Autosomal Recessive | AR |
| X-Linked | XL |
| Mitochondrial | MT |
Diseases
| Name | Inheritance | OMIM ID |
|---|---|---|
| Nemaline Myopathy 1 | AR, AD | 609284 |
| Nemaline Myopathy 10 | AR | 616165 |
| Nemaline Myopathy 2 | AR | 256030 |
| Nemaline Myopathy 3 | AR, AD | 161800 |
| Nemaline Myopathy 4 | AD | 609285 |
| Nemaline Myopathy 5 | AR | 605355 |
| Nemaline Myopathy 6 | AD | 609273 |
| Nemaline Myopathy 7 | AR | 610687 |
| Nemaline Myopathy 8 | AR | 615348 |
| Nemaline Myopathy 9 | AR | 615731 |
Related Tests
CONTACTS
Genetic Counselors
- Genetic Counselor Team - clinicaldnatesting@preventiongenetics.com
Geneticist
- Angela Gruber, PhD - angela.gruber@preventiongenetics.com
Citations
- Anderson S.L. et al. 2004. Human Genetics. 115: 185-90. PubMed ID: 15221447
- Johnston J.J. et al. 2000. American Journal of Human Genetics. 67: 814-21. PubMed ID: 10952871
- Laing N.G. et al. 2009. Human Mutation. 30: 1267-77. PubMed ID: 19562689
- Lehtokari V.L. et al. 2006. Human Mutation. 27: 946-56. PubMed ID: 16917880
- Lehtokari V.L. et al. 2014. Human Mutation. 35: 1418-26. PubMed ID: 25205138
- Leiden Muscular Dystrophy Pages- ACTA1
- North K, Ryan MM. 2015. Nemaline Myopathy. 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: 20301465
- Nowak K.J. et al. 2015. European Journal of Human Genetics. 0: N/A. PubMed ID: 25712079
- Ravenscroft G. et al. 2013. American Journal of Human Genetics. 93: 6-18. PubMed ID: 23746549
- Ryan M.M. et al. 2001. Annals of Neurology. 50: 312-20. PubMed ID: 11558787
- Ryan M.M. et al. 2003. Neurology. 60: 665-73. PubMed ID: 12601110
Order Kits
TEST METHODS
- NextGen Sequencing using PG-Select Capture Probes
- Deletion/Duplication Testing via Array Comparative Genomic Hybridization
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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