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RPE65-Associated Disorders via RPE65 Gene Sequencing with CNV Detection

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

Sequencing and Del/Dup via NGS

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
7547 RPE65$640.00 81406,81479 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 20 days.

Clinical Sensitivity

A preliminary assessment with 176 probands from 9 different countries suggested that few patients affected with Leber Congenital Amaurosis (LCA) exhibit pathogenic variants in RPE65, indicating that LCA is extremely genetically heterogeneous (Lotery et al. 2000. PubMed ID: 10766140). In another study, RPE65 pathogenic variants accounted for ~2% (3 out of 162 cases) of recessive or isolate Retinitis Pigmentosa and ~16% (7 of 45) of LCA cases (Morimura et al. 1998. PubMed ID: 9501220). Thus far, only one gross deletion has been reported involving RPE65 (Al-Gazali and Ali. 2010. PubMed ID: 20437613).

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Clinical Features

Leber Congenital Amaurosis 2 (LCA2) or Severe Early Childhood-Onset Retinal Dystrophy (SECORD) and Retinitis Pigmentosa (RP20) associated with RPE65 pathogenic variants are due to dysfunction and degeneration of photoreceptors (Cideciyan et al. 2013. PubMed ID: 23341635; Weleber et al. 2011. PubMed ID: 20811047). All affected patients have night blindness, a typical and early symptom. LCA2 or SECORD and RP20 are characterized by profound vision loss at birth, variable degrees of nystagmus, high hypermetropia, photodysphoria, oculodigital signs, keratoconus, cataracts and variable appearance to the fundus (Chung and Traboulsi. 2009. PubMed ID: 20006823). Ophthalmoscopy reveals attenuated vessels, atrophy of the optic disc and many whitish dots due to extensive retinal pigment epithelium (RPE) defects (Gu et al. 1997. PubMed ID: 9326941). The estimated prevalence of LCA is 2-3 per 100,000 live births and accounts for 10-18% of congenital blindness (Fazzi et al. 2003. PubMed ID: 12615170). Several clinical features of LCA overlap with those of RP (Perrault et al. 1996. PubMed ID: 8944027; Daiger et al. 2007. PubMed ID: 17296890; Gu et al. 1997. PubMed ID: 9326941). Both LCA and RP are clinically and genetically heterogeneous.

Genetics

LCA is inherited as an autosomal recessive trait in the vast majority of patients, while RP is either sporadic or familial with various modes of Mendelian, mitochondrial or oligogenic inheritance. To date, 14 and 25 genes have been implicated in LCA and autosomal recessive RP (AR-RP), respectively (den Hollander et al. 2008. PubMed ID: 18632300; Daiger et al. 2007. PubMed ID: 17296890). Of note, a pathogenic missense variant in RPE65 was identified by whole-exome sequencing in an autosomal dominant retinitis pigmentosa (with choroidal involvement) affected large Irish family (Bowne et al. 2011. PubMed ID: 21654732). Also, some RPE65 null allele heterozygous carriers may manifest visual symptoms (Felius et al. 2002. PubMed ID: 11786058). The clinical overlap between LCA and RP is illustrated by the involvement of six genes in both conditions. These include RPE65 (Marlhens et al. 1997. PubMed ID: 9326927; Gu et al. 1997. PubMed ID: 9326941), which encodes retinol isomerase, expressed in retinal pigment epithelium (RPE). Retinol isomerase is involved in vitamin A metabolism in the visual cycle to synthesize 11-cis-retinaldehyde. Pathogenic variants in this gene lead to no chromophore production and affect the visual pigments (Weleber et al. 2011. PubMed ID: 20811047).

RPE65 pathogenic variants account for approximately 8% of LCA and have been shown to be influenced by genetic background, environment and other factors (Li et al. 2009. PubMed ID: 18936139). About 30 different RPE65 pathogenic variants have been reported each in patients with LCA2 and RP20. LRAT, RPE65 and RDH12 pathogenic variants lead to similar phenotypes, suggesting the need to screen all these genes systematically (Sénéchal et al. 2006. PubMed ID: 17011878; Weleber et al. 2013. PubMed ID: 20301475). RPE65 pathogenic variants include missense, nonsense, splicing, and small deletions/insertions (mostly frameshift); they are distributed throughout the entire coding region.

Testing Strategy

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. All reported pathogenic, likely pathogenic, and 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.

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 RPE65 gene, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.

Indications for Test

Patients with LCA and AR-RP and family members of patients who have known pathogenic variants are candidates.

Gene

Official Gene Symbol OMIM ID
RPE65 180069
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Leber Congenital Amaurosis 2 AR 204100
Retinitis Pigmentosa 20 AR, AD 613794

Related Tests

Name
Alstrom Syndrome via ALMS1 Gene Sequencing with CNV Detection
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Autosomal Dominant Retinitis Pigmentosa Sequencing Panel with CNV Detection
Autosomal Recessive Retinitis Pigmentosa Sequencing Panel with CNV Detection
Comprehensive Inherited Retinal Dystrophies (includes RPGR ORF15) Sequencing Panel with CNV Detection
Congenital Stationary Night Blindness Sequencing Panel with CNV Detection
Focused Inherited Retinal Disorders Sequencing Panel with CNV Detection
Leber congenital amaurosis 14 (LCA14) or Early Onset Retinal Dystrophy (EORD) and Juvenile Retinitis pigmentosa via the LRAT Gene
Leber Congenital Amaurosis Sequencing Panel with CNV Detection
Retinitis Pigmentosa (includes RPGR ORF15) Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Al-Gazali and Ali. 2010. PubMed ID: 20437613
  • Bowne et al. 2011. PubMed ID: 21654732
  • Chung and Traboulsi. 2009. PubMed ID: 20006823
  • Cideciyan et al. 2013. PubMed ID: 23341635
  • Daiger et al. 2007. PubMed ID: 17296890
  • den Hollander et al. 2008. PubMed ID: 18632300
  • Fazzi et al. 2003. PubMed ID: 12615170
  • Felius et al. 2002. PubMed ID: 11786058
  • Gu et al. 1997. PubMed ID: 9326941
  • Li et al. 2009. PubMed ID: 18936139
  • Lotery et al. 2000. PubMed ID: 10766140
  • Marlhens et al. 1997. PubMed ID: 9326927
  • Morimura et al. 1998. PubMed ID: 9501220
  • Perrault et al. 1996. PubMed ID: 8944027
  • Sénéchal et al. 2006. PubMed ID: 17011878
  • Weleber et al. 2011. PubMed ID: 20811047
  • Weleber et al. 2013. PubMed ID: 20301475
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TEST METHODS

Sequencing and Deletion/Duplication Testing via NextGen Sequencing using PG-Select Capture Probes

Test Procedure

NextGen Sequencing

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.

Deletion and Duplication Testing via NGS

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing 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, PCR or qPCR before they are reported.
Analytical Validity

NextGen Sequencing

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.

Deletion and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
Analytical Limitations

NextGen Sequencing

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 and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g. pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g. single vs. two or more exons), and inadequate coverage. 
 
Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect minor CNVs, due for example to somatic mosaicism is limited.
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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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