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Congenital Fibrosis of Extraocular Muscles (Ocular Motility Disorder) Type 3A via the TUBB3 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
2019 TUBB3$540.00 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

Predicting clinical sensitivity for the TUBB3 gene is challenging due to genetic heterogeneity of Congenital fibrosis of extraocular muscles. However, analytical sensitivity should be high as all the reported variants are detectable by sequencing. TUBB3 mutation screening in 6 unrelated individuals with sporadic CFEOM3A identified de novo pathogenic variants in all the affected patients (Tischfield et al. 2010).

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Clinical Features

Congenital fibrosis of extraocular muscles (CFEOM) is an eye movement disorder characterized mainly by non-progressive, restrictive ophthalmoplegia of the extraocular muscles (EOM) and congenital blepharoptosis (Heidary et al. 2008). Congenital fibrosis of extraocular muscles (CFEOM) is a heterogeneous disorder. Depending on the gene, the disease has been categorized into different types. CFEOM1 (due to KIF21A variants), CFEOM2 (due to ARIX variants), CFEOM3A (due to TUBB3 variants), CFEOM3B (due to KIF21A variants), and CFEOM4, CFEOM5 (no genes identified yet) (Oystreck et al. 2011; Traboulsi 2004).

CFEOM3A affected patients may have additional neurological signs or symptoms such as intellectual disability, social disability, facial weakness, and progressive sensorimotor axonal polyneuropathy (Andrews et al. 1993).

Genetics

CFEOM3A is an autosomal dominant disorder, which is due to heterozygous variants in TUBB3. TUBB3 encodes neuron-specific beta-tubulin subunit,  which is required for axon guidance and maintenance in mammals (Poirier et al. 2010; Tischfield et al. 2010). In vitro studies using yeast have shown that the TUBB3 causative variants can impair tubulin heterodimer formation and disrupt the interaction of microtubules with kinesin motors. This in turn leads to innervation abnormalities in the EOM during the development of oculomotor and/or trochlear nerves (Tischfield et al. 2010). CFEOM3A is variable, asymmetrical, and variably penetrant (~90%) (Doherty et al 1999). A mutation screen involving 29 unrelated families revealed that 13 had eight TUBB3 pathogenic variants and all occurred de novo (Tischfield et al. 2010). Severe phenotype has been reported in patients with de novo mutations in TUBB3 (Poirier et al. 2010). So far only missense variants have shown to be causative for TUBB3-associated disorders (Human Gene Mutation Database).

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of all coding exons and ~ 20 bp of flanking noncoding sequence of TUBB3. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.

Indications for Test

Patients with congenital abnormalities of eye movements are candidates ((Oystreck et al. 2011; http://novel.utah.edu/diseases/rare-registry/view/Unusual_Congenital_Ocular_Motility_Disorders).

Gene

Official Gene Symbol OMIM ID
TUBB3 602661
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Andrews CV et al. 2011. Congenital Fibrosis of the Extraocular Muscles. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301522
  • Doherty EJ. et al. 1999. Investigative ophthalmology & visual science. 40: 1687-94. PubMed ID: 10393037
  • Heidary et al. 2008. Semin Ophthalmol 23: 3–8. PubMed ID: 18214786
  • Human Gene Mutation Database (Bio-base).
  • NANOS Collection of Unusual Congenital Ocular Motility Disorders and Strabismus
  • Oystreck DT. et al. 2011. Journal of neuro-ophthalmology : the official journal of the North American Neuro-Ophthalmology Society. 31: 69-77.  PubMed ID: 21317732
  • Poirier K. et al. 2010. Human molecular genetics. 19: 4462-73.  PubMed ID: 20829227
  • Tischfield MA. et al. 2010. Cell. 140: 74-87.  PubMed ID: 20074521
  • Traboulsi EI. 2004. Transactions of the American Ophthalmological Society. 102: 373-89. PubMed ID: 15747768
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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.

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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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