Focused Inherited Retinal Disorders Sequencing Panel with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||CPT Code Copy CPT Codes|
|Full Panel Price*||$640|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|7537||Genes x (31)||$640||81404(x3), 81406(x2), 81408(x2), 81479(x55)||Add|
We are happy to accommodate requests for testing single genes in this panel or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available.
This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
Clinical sensitivity for the AIPL1 (4-8%), CEP290 (20%), CRB1 (10%), CRX (3%), GUCY2D (21%), LCA5 (1-2%), RDH12 (4%), RPE65 (3-16%), RPGRIP1 (5%), TULP1 (1.7%) genes is known (Weleber et al. 2013). The most common genes involved in autosomal recessive (AR) Retinitis Pigmentosa (RP) are USH2A (10-15%), PDE6A (2-5%), PDE6B (2-5%), RPE65 (2-5%), and CNGA1 (1-2%). PCARE and RHO each account for less than 1% of RP cases (Fahim et al. 2013. PubMed ID: 20301590). The most common genes involved in autosomal dominant (AD) RP are RHO (26-28% of RP cases), RP1 (6%), NR2E3 (0.5-1.5%), and PRPF8/RP13 (3%) (Fahim et al. 2013. PubMed ID: 20301590; Daiger et al. 2010. PubMed ID: 20238032; Sullivan et al. 2013. PubMed ID: 23950152). However, for other genes, due to the limited number of cases, estimation of clinical sensitivity is difficult (Hanein et al. 2004. PubMed ID: 15024725; Weleber et al. 2013. PubMed ID: 20301475). Together these genes account for 70-75% of the Leber Congenital Amaurosis cases (Wang et al. 2015. PubMed ID: 26047050; den Hollander et al. 2008. PubMed ID: 18632300).
A study by Perrault et al. (2000) identified two gross deletions and one duplication in GUCY2D out of 118 patients affected with Leber Congenital Amaurosis (Perrault et al. 2000. PubMed ID: 10951519). Copy number variants have also been reported in CEP290, CHM, CRB1, CRX, EYS, GUCY2D, LCA5, MERTK, NMNAT1, PDE6B, PRPF8, RDH12, RHO, RPE65, RPGRIP1, TULP1 and USH2A (Human Gene Mutation Database).
Inherited retinal disorders (IRD) are the leading cause of blindness in the western world (1 in 3000 people). Identifying the genetic cause for the IRD is challenging due to genetic heterogeneity. According to the World Health Organization (WHO) and the American Academy of ophthalmology (AAO), ~ 80% of blindness can be prevented or cured or the disease progression could be slowed if detected at early stages. Given these statistics, the importance of early and accurate diagnosis cannot be understated. Currently, molecular diagnosis of the IRD is gaining importance due to the emerging treatments such as gene therapy (Sahel et al. 2014. PubMed ID: 25324231; Chen et al. 2013. PubMed ID: 23661368).
Identifying the genetic cause for the IRD is challenging due to genetic heterogeneity. So far, ~300 loci have been mapped and over 250 genes have been identified to be associated with retinal disorders (RetNet). This panel tests AIPL1, PCARE (C2orf71), CABP4, CEP290, CHM, CNGA1, CRB1, CRX, EYS, GUCY2D, IMPDH1, IQCB1, KCNJ13, LCA5, LRAT, NMNAT1, NR2E3, OTX2, PDE6A, PDE6B, PRPF8, RD3, RDH12, RDH5, RHO, RP1, RPE65, RPGRIP1, SPATA7, TULP1 and USH2A.
Pathogenic variants in AIPL1, CABP4, CEP290, CNGA1, EYS, IQCB1, LCA5, LRAT, NMNAT1, PCARE, PDE6A, RD3, RPGRIP1, SPATA7, TULP1 and USH2A cause autosomal recessive (AR) retinal disorders (Chen et al. 2013. PubMed ID: 23661368). Pathogenic variants in PRPF8, and OTX2 cause autosomal dominant (AD) retinal disorders (Bowne et al. 2006. PubMed ID: 16384941; Henderson et al. 2009. PubMed ID: 19956411; Swaroop et al. 1999. PubMed ID: 9931337; Zhao et al. 2006. PubMed ID: 16612614). CRB1, CRX, GUCY2D, IMPDH1, PDE6B, RDH5, RDH12, RP1, KCNJ13, RHO, NR2E3 and RPE65 are implicated in both AD and AR retinal disorders (Kohl et al. 2012. PubMed ID: 22901948; Piri et al. 2005. PubMed ID: 15629837; Wang et al. 2013. PubMed ID: 23847139; Weleber et al. 1993. PubMed ID: 8240110; Udar et al. 2003. PubMed ID: 12552567; Hanein et al. 2002. PubMed ID: 12325031; McKay et al. 2005. PubMed ID: 15623792; Abouzeid et al. 2006. PubMed ID: 16543197; Swaroop et al. 1999. PubMed ID: 9931337. 1999; Hejtmancik et al. 2008. PubMed ID: 18179896). Pathogenic variants in CHM are associated with X-linked choroideremia (van den Hurk et al. 2003. PubMed ID: 12827496). Pathogenic variants in these genes also cause other retinal disorders (Weleber. 2002. PubMed ID: 12187427; Wang et al. 2015. PubMed ID: 26047050; Wang et al. 2013. PubMed ID: 23847139; Online Mendelian Inheritance in Man; Human Gene Mutation Database).
See individual gene test descriptions for information on molecular biology of gene products and spectra of pathogenic variants.
For this Next Generation Sequencing (NGS) test, 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 regions not captured or with insufficient number of sequence reads.
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.
Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.
This panel provides 100% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.
Indications for Test
Candidates for this test are inherited retinal disorders patients, family members of patients who have known pathogenic variants and carrier testing for at-risk family members.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - firstname.lastname@example.org
- Madhulatha Pantrangi, PhD - email@example.com
- Abouzeid et al. 2006. PubMed ID: 16543197
- Bowne et al. 2006. PubMed ID: 16384941
- Chen et al. 2013. PubMed ID: 23661368
- Daiger et al. 2010. PubMed ID: 20238032
- den Hollander et al. 2008. PubMed ID: 18632300
- Fahim et al. 2013. PubMed ID: 20301590
- Hanein et al. 2002. PubMed ID: 12325031
- Hanein et al. 2004. PubMed ID: 15024725
- Hejtmancik et al. 2008. PubMed ID: 18179896
- Henderson et al. 2009. PubMed ID: 19956411
- Human Gene Mutation Database (Bio-base).
- Kohl et al. 2012. PubMed ID: 22901948
- McKay et al. 2005. PubMed ID: 15623792
- Perrault et al. 2000. PubMed ID: 10951519
- Piri et al. 2005. PubMed ID: 15629837
- RetNet: Genes and Mapped Loci Causing Retinal Diseases
- Sahel et al. 2014. PubMed ID: 25324231
- Sullivan et al. 2013. PubMed ID: 23950152
- Swaroop et al. 1999. PubMed ID: 9931337
- Udar et al. 2003. PubMed ID: 12552567
- van den Hurk et al. 2003. PubMed ID: 12827496
- Wang et al. 2013. PubMed ID: 23847139
- Wang et al. 2015. PubMed ID: 26047050
- Weleber et al. 1993. PubMed ID: 8240110
- Weleber et al. 2013. PubMed ID: 20301475
- Weleber. 2002. PubMed ID: 12187427
- Zhao et al. 2006. PubMed ID: 16612614
Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 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.
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 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.
Deletion and Duplication Testing via NGS
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.
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 ~10 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.
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.
- 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.
(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.
(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.
(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.