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Congenital Myasthenic Syndromes and Lethal Multiple Pterygium Syndrome via the CHRNA1 Gene

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

Sequencing

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
401 CHRNA1$680.00 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

Sensitivity for CMS testing is at least 50% overall; 30% for CHRNE, 10% for RAPSN, and 7.5% for COLQ (GeneReviews, Abicht and Lochmüller, 2006).  CHRNA1 is a rare cause of CMS, and too few cases of LMPS have been reported to estimate sensitivity.

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Deletion/Duplication Testing via aCGH

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
600 CHRNA1$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

Congenital myasthenic syndromes (CMS) are disorders of the neuromuscular junction resulting from abnormalities of presynaptic, synaptic, or post synaptic proteins. CMS are characterized by fatigable weakness affecting limb, ocular, facial, and bulbar muscles. Neonates present with feeding problems, choking, feeble cry, and muscle weakness. Patients presenting in later childhood are seen with abnormal exercise-induced fatigue and difficulty running. Most patients present prior to 2 years of age although rare exceptions are reported (eg. Croxen et al. Neurol 59:162-168, 2002). Symptoms are extremely variable, and are in some case induced by febrile illness, infection, or excitement (eg. Byring et al. Neuromuscul Disord 12:548-553, 2002). Life threatening respiratory crises may occur in affected neonates or older children. CMS may be differentiated from myasthenia gravis, an acquired autoimmune disorder, by earlier age at onset and by negative serology tests for anti-acetylcholine receptor (AchR) and anti-Musk antibodies. Lethal multiple pterygium syndrome (LMPS, OMIM 253290) is characterized by prenatal growth deficiency, contractures, pterygia, and dysmorphic facies. Joint contractures and multiple pterygia are universal findings. Pulmonary hypoplasia is likely the primary cause of mortality. Contractures are found at the elbows, knees, hips, shoulders, hands, and feet. Pterygia are found between the chin and sternum as well as the popliteal, axillary, antecubital, and ankle areas. Facial features include ocular hypertelorism, epicanthal folds, small chin and mouth, and low-set ears.

Genetics

Abnormalities of proteins involved with neuromuscular transmission underlie (Congenital Myasthenic Syndrome (CMS), limb girdle CMS, Pena-Shokeir syndrome, and multiple pterygium syndromes. These disorders, which may represent a phenotypic continuum of a single entity, are most often inherited in an autosomal recessive manner. CHRNA1 gene mutations have been found in patients with fast and slow channel CMS (Engel and Sine Curr Opinion in Pharmacol 5:308-321, 2005). Fast channel CMS (OMIM 608930) secondary to loss-of-function mutations in the acetylcholine receptor alpha gene (CHRNA1, OMIM 100690) is mostly inherited as a recessive condition (Ohno et al. Neuron 17:157-170, 1996; Wang et al. Nature Neurosci 2:226-233, 1999). However, dominant inheritance of CHRNA1-related fast channel CMS has been reported (Webster et al. Neurol 62:1090-1096, 2004). Slow-channel CMS (OMIM 601462) secondary to gain-of-function mutations in the CHRNA1 gene is inherited as a dominant condition (Engel et al. Hum Molec Genet 5:1217-1227, 1996; Shen et al. Ann Neurol 60:128-136, 2006). Lethal multiple pterygium syndrome is inherited as an autosomal recessive condition (Michalk et al. Am J Hum Genet 82:464-476, 2008). Null and low expressor CHRNA1 mutations in the homozygous or compound heterozygous state lead to CMS with end-plate AChR deficiency (Engel and Sine 2005).

Testing Strategy

The alpha subunit of the acetylcholine receptor is encoded by exons 1 – 10 of the CHRNA1 gene located on chr 2q24. Testing is accomplished by amplifying the coding exons 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

A comprehensive diagnostic algorithm can be found in (GeneReviews, Abicht and Lochmüller, 2006).

Gene

Official Gene Symbol OMIM ID
CHRNA1 100690
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Congenital Myasthenic Syndrome Sequencing Panel
Fetal Akinesia Deformation Sequence/Lethal Multiple Pterygium Syndrome Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Angela Abicht, Hanns Lochm?ller (2006). "Congenital Myasthenic Syndromes."
  • Angela Abicht, Hanns Lochmuller (2006). "Congenital Myasthenic Syndromes."
  • Byring RF, Pihko H, Tsujino A, Shen XM, Gustafsson B, Hackman P, Ohno K, Engel AG, Udd B. 2002. Congenital myasthenic syndrome associated with episodic apnea and sudden infant death. Neuromuscul Disord 12: 548-553. PubMed ID: 12117478
  • Croxen R, Hatton C, Shelley C, Brydson M, Chauplannaz G, Oosterhuis H, Vincent A, Newsom-Davis J, Colquhoun D, Beeson D. 2002. Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes. Neurology 59: 162-168. PubMed ID: 12141316
  • Engel, A. G., et.al. (1996). "New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome." Hum Mol Genet 5(9): 1217-27. PubMed ID: 8872460
  • Engel, A. G., Sine, S. M. (2005). "Current understanding of congenital myasthenic syndromes." Curr Opin Pharmacol 5(3): 308-21. PubMed ID: 15907919
  • Michalk, A., et.al. (2008). "Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders." Am J Hum Genet 82(2): 464-76. PubMed ID: 18252226
  • Ohno, K., et.al. (1996). "Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor epsilon subunit." Neuron 17(1): 157-70. PubMed ID: 8755487
  • Shen, X. M., et.al. (2006). "Slow-channel mutation in acetylcholine receptor alphaM4 domain and its efficient knockdown." Ann Neurol 60(1): 128-36. PubMed ID: 16685696
  • Wang, H. L., et.al. (1999). "Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating." Nat Neurosci 2(3): 226-33. PubMed ID: 10195214
  • Webster, R., et.al. (2004). "Mutation in the AChR ion channel gate underlies a fast channel congenital myasthenic syndrome." Neurology 62(7): 1090-6. PubMed ID: 15079006
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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.

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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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