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NDP-Related Vitreoretinopathies via the NDP 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
1067 NDP$440.00 81404 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

According to Riveiro-Alvarez et al., ~ 85% of the patients clinically diagnosed with Norrie Disease have point mutations in NDP gene (Riveiro-Alvarez et al. 2005). A molecular analysis in a four generation FEVR family revealed a missense mutation c.370C>T (Leu124Phe) in the highly conserved region of the NDP gene. It was detected in all of the affected males, but not in the unaffected family members or in normal controls (Chen et al. 1993).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 NDP$690.00 81403 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

NDP-related vitreoretinopathies are characterized by a spectrum of retinal pathology that occurs at birth and has varying degrees of severity.

The most severe form is Norrie Disease (ND), which is a rare disorder characterized by congenital blindness due to severe retinal dysgenesis. One third of ND affected individuals develop hearing loss and about one half exhibit intellectual disability (Ott et al. 2000). In some ND cases, more complex phenotypes such as microphthalmia, growth failure, and seizures are present. The other less severe phenotypes of NDP-related vitreoretinopathies include familial exudative vitreoretinopathy (FEVR), Coats disease, retinopathy of prematurity (ROP) and persistent hyperplastic primary vitreous (PHPV) (Sims 2009).

FEVR is characterized by abnormal vascularisation of the peripheral retina. FEVR penetrance is reported to be 100% and shows an extremely variable clinical expression, even within a family and is clearly asymmetric. 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).

Coats' disease is characterized by abnormal development of the retinal vessels (telangiectasis) which leads to the massive intraretinal and subretinal lipid deposition that in turn results in exudative retinal detachment. The classic form of Coats' disease is always isolated, unilateral and affects males. Often, it is difficult to differentiate advanced Coats' disease from retinoblastoma on ophthalmoscopic findings alone (Haik 1991).

Retinopathy of prematurity (ROP) is a multifactorial disease, which is characterized by complication of low gestational age and low birth weight. Early detection is very important in order to identify the independent risk factors for the development of ROP, which may lead to blindness (Delport et al. 2002; Mathew et al. 2002).

PHPV, also known as persistent fetal vasculature, is a rare congenital developmental malformation of the eye with an estimated prevalence ranging from approximately one in 2000 to one in 9000 patients. Bilaterality occurs in 9–17% of these cases. PHPV is characterized by a fibrovascular stalk that extends from the optic disc to the lens (Makino et al. 2013; Sims 1993; Shastry 2009).

Genetics

All NDP-related vitreo-retinopathies are associated with mutations in the NDP gene, which is located on chromosome Xp11.4 and exhibits X-linked mode of inheritance. Approximately 95% of affected males have disease causing sequence variations in NDP (Sims 2009). Female carriers are often asymptomatic, but in rare cases may have some disease phenotype due to  nonrandom X inactivation (Sims et al. 1997). So far, about 150 pathogenic sequence variations (including missense, nonsense, splicing,regulatory, small and gross insertions and deletions) in NDP have been associated with NDP-related vitreoretinopathies (Human Gene Mutation Database).

NDP encoded protein Norrin is a member of the cystine knot growth factor family and is a major regulator in the formation of the retinal vasculature during eye development (Ohlmann and Tamm 2012). It also protects retinal ganglion cells from oxygen-induced retinal vascular damage. Norrin is a retinal signaling molecule and is constitutively expressed in Müller cells of the eye. It specifically binds to Frizzled-4 (FZD4) receptors and activates the classic Wnt/β-catenin signaling pathway, which in turn induces neuroprotective effects of Norrin (Seitz et al. 2010; Braunger et al. 2013). Perturbations in the Wnt pathway have been associated with several retinal disorders (Lad et al. 2009).

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of all coding exons (2-3) of the NDP gene. The full coding region of each exon plus ~20 bp of flanking non-coding DNA on either side are sequenced. This test also covers the five noncoding NDP variants (c.-208+1G>A; c.-208+2T>G; c.-208+5G>A; c.-207-1G>A; c.*715T>C) that have been reported to be pathogenic (Human Gene Mutation Database). We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with a known mutation or to confirm research results.

Indications for Test

All patients with symptoms suggestive of NDP-related vitreoretinopathies (described in the clinical features section), and relatives of patients with known NDP mutations.

Gene

Official Gene Symbol OMIM ID
NDP 300658
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Test

Name
Vitreoretinopathy Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Braunger BM, Ohlmann A, Koch M, Tanimoto N, Volz C, Yang Y, Bösl MR, Cvekl A, Jägle H, Seeliger MW, Tamm ER. 2013. Constitutive overexpression of Norrin activates Wnt/β-catenin and endothelin-2 signaling to protect photoreceptors from light damage. Neurobiol. Dis. 50: 1–12. PubMed ID: 23009755
  • Chen ZY, Battinelli EM, Fielder A, Bundey S, Sims K, Breakefield XO, Craig IW. 1993. A mutation in the Norrie disease gene (NDP) associated with X-linked familial exudative vitreoretinopathy. Nat. Genet. 5: 180–183. PubMed ID: 8252044
  • Delport SD, Swanepoel JC, Odendaal PJL, Roux P. 2002. Incidence of retinopathy of prematurity in very-low-birth-weight infants born at Kalafong Hospital, Pretoria. S. Afr. Med. J. 92: 986–990. PubMed ID: 12561416
  • Haik BG. 1991. Advanced Coats’ disease. Trans Am Ophthalmol Soc 89: 371–476. PubMed ID: 1808814
  • Human Gene Mutation Database (Bio-base).
  • Lad EM, Cheshier SH, Kalani MYS. 2009. Wnt-signaling in retinal development and disease. Stem Cells Dev. 18: 7–16. PubMed ID: 18690791
  • Makino S, Ohkubo Y, Tampo H. 2013. Prepapillary Vascular Loop Associated with Persistent Hyperplastic Primary Vitreous. Case Reports in Ophthalmological Medicine 2013: 1–2. PubMed ID: 23762694
  • Mathew MRK, Fern AI, Hill R. 2002. Retinopathy of prematurity: are we screening too many babies? Eye (Lond) 16: 538–542. PubMed ID: 12194065
  • Ohlmann A, Tamm ER. 2012. Norrin: molecular and functional properties of an angiogenic and neuroprotective growth factor. Prog Retin Eye Res 31: 243–257. PubMed ID: 22387751
  • Ott S, Patel RJ, Appukuttan B, Wang X, Stout JT. 2000. A novel mutation in the Norrie disease gene. J AAPOS 4: 125–126. PubMed ID: 10773814
  • Riveiro-Alvarez R. et al. 2005. Molecular Vision. 11: 705-12.  PubMed ID: 16163268
  • Seitz R, Hackl S, Seibuchner T, Tamm ER, Ohlmann A. 2010. Norrin Mediates Neuroprotective Effects on Retinal Ganglion Cells via Activation of the Wnt/ -Catenin Signaling Pathway and the Induction of Neuroprotective Growth Factors in Muller Cells. Journal of Neuroscience 30: 5998–6010. PubMed ID: 20427659
  • Shastry BS. 2009. Persistent hyperplastic primary vitreous: congenital malformation of the eye. Clin. Experiment. Ophthalmol. 37: 884–890. PubMed ID: 20092598
  • Sims KB, Irvine AR, Good WV. 1997. Norrie disease in a family with a manifesting female carrier. Arch. Ophthalmol. 115: 517–519. PubMed ID: 9109762
  • Sims KB. 2009. NDP-Related Retinopathies. 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: 20301506
  • Toomes C. et al. 2004. American Journal of Human Genetics. 74: 721-30. PubMed ID: 15024691
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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