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Familial Exudative Vitreoretinopathy 5 (FEVR5) via the TSPAN12 Gene

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1069 TSPAN12$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

Mutations in the TSPAN12 gene account for 3-10% of familial exudative vitreoretinopathy cases (Poulter et al. 2010; Toomes and Downey 2011).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 TSPAN12$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

Familial exudative vitreoretinopathy (FEVR) is an inherited ocular disorder characterized by abnormal vascularisation of the peripheral retina. FEVR penetrance is reported to be close to 100%, but shows a variable clinical expression, even within families. At the milder end of the disease spectrum, individuals are asymptomatic or may have a small area of avascularity in the peripheral retina, whereas at the severe end, individuals are legally blind during the first decade of life (Toomes et al. 2004). The secondary retinal pathologies include retinal folds and detachment in association with retinal traction, subretinal or intraretinal exudation, and fibrovascular proliferation at the junction between vascularised and non-vascularised retina. Rarely retinoschisis and giant retinal tears have been reported (Toomes and Downey 2011).

Genetics

FEVR has been linked to five different loci (EVR1 to EVR5). EVR1, EVR3 and EVR4 are located on 11q13–23; EVR2 is on X-chromosome and EVR5 is on chromosome 7. The EVR4 locus has been reported as extremely rich in genes that are associated with a range of retinal disorders like Best disease (BEST1), oculocutaneous albinism (TYR), retinitis pigmentosa (ROM1), Usher’s syndrome (MYO7A), Bardet Biedel syndrome (BBS1) and inflammatory vitreoretinopathy (CAPN5) (Bamashmus et al. 2000; Toomes et al. 2004b). FEVR is genetically heterogeneous and exhibits autosomal dominant (ad), autosomal recessive (ar) and X-linked inheritance (XL) with ad being the most common mode. So far four causative genes, LRP5 (low-density lipoprotein 5), FZD4 (frizzled 4), NDP (norrin), and TSPAN12 (tetraspanin-12,) have been identified. The proteins encoded by these genes act as ligand and receptors in Wnt signaling pathway, which is highly conserved among species and plays an important  role in eye organogenesis and angiogenesis (Warden et al. 2007; Nikopoulos et al. 2010). Together, all four genes are responsible for only 50% of the FEVR cases, indicating the significant genetic heterogeneity of FEVR, and that additional FEVR genes involved in Wnt signaling pathway remain to be identified (Poulter et al. 2010).

TSPAN12, which is located on chromosome 7, encodes the protein tetraspanin that belongs to the 33 member four-transmembrane-domain protein superfamily (also referred to as tetraspans or TM4SF proteins). These proteins have the ability to interact with each other and also with other proteins to form large multimolecular membrane complexes called tetraspanin webs (Poulter et al. 2010; Hemler 2005). These transmembrane proteins have been implicated in a diverse range of biological processes, including signal transduction, metastasis, cell proliferation, differentiation and migration (Poulter et al. 2010; Berditchevski 2001; Yunta and Lazo 2003; Levy and Shoham 2005). TSPAN12 expression is shown to be restricted to the retinal vasculature and is required for retinal vascular development. TSPAN12 enhances Norrin/β-Catenin Signaling in retinal endothelial cells (Junge et al. 2009). TSPAN12 mutations are associated with both adFEVR and arFEVR. However, mutations in both alleles of TSPAN12 have been reported to cause severe form of FEVR or retinal dysplasia (Poulter et al. 2012). About 20 mutations in TSPAN12 have been reported to cause FEVR and include missense, nonsense, splicing, small deletions, a small insertion and one gross deletion (Human Gene Mutation Database).

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of all coding exons (2-8) of the TSPAN12 gene. The full coding region of each exon plus ~20 bp of flanking non-coding DNA on either side are sequenced. 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

All patients with symptoms suggestive of familial exudative vitreoretinopathy (FEVR), and relatives of patients with known TSPAN12 mutations.

Gene

Official Gene Symbol OMIM ID
TSPAN12 613138
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Exudative Vitreoretinopathy 5 613310

Related Test

Name
Vitreoretinopathy Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bamashmus MA, Downey LM, Inglehearn CF, Gupta SR, Mansfield DC. 2000. Genetic heterogeneity in familial exudative vitreoretinopathy; exclusion of the EVR1 locus on chromosome 11q in a large autosomal dominant pedigree. Br J Ophthalmol 84: 358–363. PubMed ID: 10729291
  • Berditchevski F. 2001. Complexes of tetraspanins with integrins: more than meets the eye. Journal of Cell Science 114: 4143–4151. PubMed ID: 11739647
  • Hemler ME. 2005. Tetraspanin functions and associated microdomains. Nat. Rev. Mol. Cell Biol. 6: 801–811. PubMed ID: 16314869
  • Human Gene Mutation Database (Bio-base).
  • Junge HJ, Yang S, Burton JB, Paes K, Shu X, French DM, Costa M, Rice DS, Ye W. 2009. TSPAN12 Regulates Retinal Vascular Development by Promoting Norrin- but Not Wnt-Induced FZD4/β-Catenin Signaling. Cell 139: 299–311. PubMed ID: 19837033
  • Levy S, Shoham T. 2005. Protein-protein interactions in the tetraspanin web. Physiology (Bethesda) 20: 218–224. PubMed ID: 16024509
  • Nikopoulos K. et al. 2010. Human Mutation. 31: 656-66. PubMed ID: 20340138
  • Poulter J.A. et al. 2010. American Journal of Human Genetics. 86: 248-53.  PubMed ID: 20159112
  • Poulter JA, Davidson AE, Ali M, Gilmour DF, Parry DA, Mintz-Hittner HA, Carr IM, Bottomley HM, Long VW, Downey LM, Sergouniotis PI, Wright GA, et al. 2012. Recessive Mutations in TSPAN12 Cause Retinal Dysplasia and Severe Familial Exudative Vitreoretinopathy (FEVR). Investigative Ophthalmology & Visual Science 53: 2873–2879. PubMed ID: 22427576
  • Toomes C, Downey L. 2011. Familial Exudative Vitreoretinopathy, Autosomal Dominant. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301326
  • Toomes C, Downey LM, Bottomley HM, Scott S, Woodruff G, Trembath RC, Inglehearn CF. 2004. Identification of a fourth locus (EVR4) for familial exudative vitreoretinopathy (FEVR). Mol. Vis. 10: 37–42. PubMed ID: 14737064
  • Toomes C. et al. 2004. American Journal of Human Genetics. 74: 721-30. PubMed ID: 15024691
  • Warden S.M. et al. 2007. Seminars in Ophthalmology. 22: 211-7.  PubMed ID: 18097984
  • Yunta M, Lazo PA. 2003. Tetraspanin proteins as organisers of membrane microdomains and signalling complexes. Cell. Signal. 15: 559–564. PubMed ID: 12681443
Order Kits
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.

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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