Forms

'Bull's Eye' Macular Dystrophy (BEM), Cone-Rod Dystrophy 12 (CORD12), Retinitis Pigmentosa 41 (RP41) and Stargardt Disease 4 (STGD4) via the PROM1 Gene

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

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
3191 PROM1$990.00 81479 Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

For Sanger Sequencing click here.
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 28 days.

Clinical Sensitivity

PROM1 mutations are a relatively rare cause of blindness. In a study done in a large family, where all six affected individuals had homozygous PROM1 mutations, five of the six unaffected family members were heterozygous carriers (Zhang et al. Hum Genet 122(3-4):293-299, 2007). Another study identified homozygous PROM1 frameshift mutations in three siblings from a consanguineous Arab family, who had prominent axial myopia (Pras et al. Mol Vis 15:1709-1716 2009). A third study done in 41 individuals from five unrelated families with autosomal dominant BEM, revealed heterozygous c.1117C>T  mutations in PROM1 in all affected family members (in family C all 8 affected individuals had this variant and unaffected individuals did not) (Michaelides et al. Invest Ophthalmol Vis Sci 51(9):4771-4780, 2010).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PROM1$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 Sensitivity

To date, no gross deletions or duplications have been reported in PROM1 (Human Gene Mutation Database).

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Clinical Features

'Bull's Eye' Macular Dystrophy or Macular Dystrophy, Retinal 2 (BEM/MCDR2; OMIM 608051) is clinically characterized by mild visual impairment that is usually evident in the first two decades of life. Most prominently, early macular abnormalities include annular retinal pigment epithelium (RPE) atrophy due to the lipofuscin accumulation, sparing the central fovea that gives the distinct BEM appearance. Other symptoms include central scotoma, and flash electroretinogram abnormalities due to cone dysfunction. As the disease progresses, more widespread rod and cone abnormalities are seen in the initially spared central fovea (Michaelides et al. Invest Ophthalmol Vis Sci 44(4):1657-1662, 2003).

Retinitis pigmentosa or rod cone dystrophies (RP/RCDs) represent a group of hereditary retinal dystrophies with a worldwide prevalence of ~1 in 4000 (Booij et al. J Med Genet 42(11): e67, 2005). RP is clinically characterized by retinal pigment deposits visible on fundus examination, night blindness, followed by progressive loss of peripheral vision in daylight, which eventually leads to blindness (van Soest et al. Surv Ophthalmol 43(4):321-34,1999).

Cone rod dystrophies (CORDs/CRDs) are 10 times less common than RP (1/40,000). They are characterized by dysfunction or degeneration of cone photoreceptors with relative preservation of rod function in the initial stages. The most common symptoms are photophobia, reduced visual acuity, and dyschromatopsia. Fundus changes may vary from mild pigment granularity to a distinct atrophic lesion in the central macula. As the disease progresses, degeneration of rod photoreceptors also occurs and leads to progressive night blindness and peripheral visual field loss (Hamel. Orphanet J Rare Dis 2:7, 2007). 

Stargardt disease (STGD) is the most commonly inherited macular dystrophy with a prevalence of ~1/10,000 and a carrier frequency of 2% (Shastry. Int J Mol Med 21(6):715-720, 2008). Clinically it is characterized by reduced central vision, bilateral, symmetric, atrophic lesions in the macula and underlying RPE and by the presence of prominent flecks in the posterior pole of the retina (Kniazeva et al. Am J Hum Genet 64(5):1394-1399, 1999).

Genetics

A pentaspan membrane glycoprotein prominin-1 (also known as CD133/AC133) encoded by PROM1 was originally classified as a cell surface marker for human hematopoietic stem/progenitor cells, though it is also expressed in some differentiated cells (Yin et al. Blood 90(12):5002-5012, 1997). Missense mutations in PROM1 have been associated with several severe forms of autosomal dominant retinal dystrophies such as BEM (Michaelides et al., 2003), CORD12 (OMIM 612657) (Yang et al. J Clin Invest 118(8):2908-2916, 2008), and STGD4 (OMIM 603786)(Kniazeva et al., 1999). Frameshift mutations in PROM1, resulting in premature stop codons and truncation of the encoded protein and most likely representing null mutations, cause autosomal-recessive RP41 (OMIM 612095) (Zhang et al. Hum Genet 122(3-4):293-299, 2007). Early and severe progressive rod and cone degeneration are the hallmark of PROM1 truncating mutations (Yang et al., 2008; Permanyer et al. Invest Ophthalmol Vis Sci 51(5):2656-63, 2010; Pras et al. Mol Vis 15(5):1709-1716, 2009). Due to alternative splicing, multiple PROM1 protein isoforms have been reported in human tissues (Yang et al., 2008; Permanyer et al., 2010). Two isoforms, s11 and s12, are most prominent in human retina, whereas the s2 isoform spans all the coding exons and is expressed faintly and other isoforms are barely detectable (Permanyer et al., 2010). In the retina, PROM1 is expressed in both rod and cone photoreceptors predominantly at the base of the photoreceptor outer segments (OSs). The photoreceptor OSs comprises a stack of over 1,000 densely packed disks that harbors rhodopsin and the phototransduction machinery. The entire OS is continually renewed by the constant formation of new disks at its base and shedding of older disks from its apex (Young et al. J Cell Biol 33(1):61-72, 1967). The PROM1 protein has prominent function in visual phototransduction and acts as a key regulator of disk morphogenesis during early retinal development (Permanyer et al., 2010).

Testing Strategy

For this Next Generation (NextGen) test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the gene 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.

Indications for Test

Patients with early and severe progressive rod and cone degeneration, particularly with high myopia are candidates for this test. Patients with symptoms consistent of BEM, CORD12, RP41 and STGD4 are also candidates.

Gene

Official Gene Symbol OMIM ID
PROM1 604365
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Autosomal Recessive Retinitis Pigmentosa Sequencing Panel with CNV Detection
Cone-Rod Dystrophy Sequencing Panel
Flecked Retina Disorder Sequencing Panel with CNV Detection
Retinitis Pigmentosa (includes RPGR ORF15) Sequencing Panel with CNV Detection
Stargardt Disease (STGD) and Macular Dystrophies (includes RPGR ORF15) Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Booij JC. 2005. Identification of mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa. Journal of Medical Genetics 42: e67–e67. PubMed ID: 16272259
  • Hamel CP. 2007. Cone rod dystrophies. Orphanet J Rare Dis 1;2:7. PubMed ID: 17270046
  • Human Gene Mutation Database (Bio-base).
  • Kniazeva, M. et al. (1999). "A new locus for autosomal dominant stargardt-like disease maps to chromosome 4." Am J Hum Genet 64(5):1394-1399. PubMed ID: 10205271
  • Michaelides, M. et al. (2003). "An autosomal dominant bull's-eye macular dystrophy (MCDR2) that maps to the short arm of chromosome 4." Invest Ophthalmol Vis Sci 44(4):1657-1662. PubMed ID: 12657606
  • Michaelides, M. et al. (2010). "The PROM1 mutation p.R373C causes an autosomal dominant bull's eye maculopathy associated with rod, rod-cone, and macular dystrophy." Invest Ophthalmol Vis Sci 51(9):4771-4780. PubMed ID: 20393116
  • Permanyer, J. et al. (2010). "Autosomal recessive retinitis pigmentosa with early macular affectation caused by premature truncation in PROM1." Invest Ophthalmol Vis Sci 51(5):2656-2663. PubMed ID: 20042663
  • Pras, E. et al. (2009). "Cone-rod dystrophy and a frameshift mutation in the PROM1 gene." Mol Vis 15:1709-1716. PubMed ID: 19718270
  • Shastry BS. 2008. Evaluation of the common variants of the ABCA4 gene in families with Stargardt disease and autosomal recessive retinitis pigmentosa. International journal of molecular medicine 21: 715–720. PubMed ID: 18506364
  • Van Soest S., Westerveld A. 1999. Survey of ophthalmology. 43: 321-34. PubMed ID: 10025514
  • Yang, Z. et al. (2008). "Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice." J Clin Invest 118(8):2908-2916. PubMed ID: 18654668
  • Yin, A.H. et al. (1997). "AC133, a novel marker for human hematopoietic stem and progenitor cells." Blood 90(12):5002-5012. PubMed ID: 9389720
  • Young, R.W. et al. (1967). "The renewal of photoreceptor cell outer segments." J Cell Biol 33(1):61-72. PubMed ID: 6033942
  • Zhang, Q. et al. (2007). "Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene." Hum Genet 122(3-4):293-299. PubMed ID: 17605048
Order Kits
TEST METHODS

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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