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RPGRIP1-Related Retinal Disorders via the RPGRIP1 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
3031 RPGRIP1$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

A mutation screening in a group of 35 unrelated patients with arRP (17 patients), LCA (9 patients), and isolated RP (9 patients) identified RPGRIP1 pathogenic variations in 6% of the patients (Booij et al. 2005). Genetic screening of 15 genes in eighty-seven unrelated Chinese patients with LCA identified RPGRIP1 pathogenic variations in 8% of their patient cohort (Li et al. 2011).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 RPGRIP1$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

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal degeneration. LCA is usually evident at birth or during the first months of life. LCA is clinically characterized by poor visual function often accompanied by nystagmus, abnormal pupillary responses, photophobia, high hyperopia, markedly diminished electroretinogram (ERG) and keratoconus condition due to oculo-digital signs of Franceschetti such as eye poking, pressing, and rubbing the eyes with a knuckle or finger (Weleber et al. 2013; Perrault et al. 1996). The estimated prevalence of LCA is 2-3 per 100,000 live births. LCA accounts for 10-18% of congenital blindness cases (Fazzi et al. 2003).

Genetics

LCA is a genetically heterogeneous disorder. To date, approximately 19 genes have been implicated in the pathogenesis of LCA (Weleber et al. 2013; Chen et al. 2013). Together these genes account for 70% of LCA cases. These genes encode proteins that have a wide range of retinal functions, such as photoreceptor morphogenesis, phototransduction, vitamin A cycling, guanine synthesis, and outer segment phagocytosis (den Hollander et al. 2008). Mutations in these genes cause not only LCA, but also other retinal distrophies (Weleber 2002). Genetic variations in RPGRIP1, which encodes the retinitis pigmentosa GTPase regulator (RPGR)-interacting protein 1, are responsible for 4.5–6% of LCA (Won et al. 2009).

RPGRIP1 protein localizes within photoreceptors, predominantly at the connecting cilia and the outer segments (OS), where RPGR protein (majorly associated with X-linked RP) also localizes (Roepman et al. 2005). RPGRIP1 interacts with RPGR, RPGRIP1L (RPGRIP1- like, a protein homolog based on 29% amino acid identity with RPGRIP1) and Nek4 (never in mitosis A (NIMA)-related proteinkinase family protein). This interaction is likely important in photoreceptors; specifically, in regulating ciliary homeostasis. It is likely that RPGRIP1 and RPGRIP1L act as cilium-specific scaffolds in recruiting Nek4 signaling network and in turn regulate cilium integrity and stability (Coene et al. 2011). It has also been reported that RPGRIP1 may be part of the multiprotein complex, which is required for disk morphogenesis, putatively by regulating actin cytoskeleton dynamics (Zhao et al. 2003). Thus far, sequence variations in RPGRIP1 appear to be typically reported in LCA patients (Koenekoop 2005). Due to its interaction with RPGR, it is conceivable that genetic defects in RPGRIP1 that compromise their interaction may cause phenotypes less severe than LCA, such as RP or CRD (cone rod dystrophy) (Cremers et al. 2002; Roepman et al. 2005).

There are about eighty documented pathogenic variations in RPGRIP1 which are associated with autosomal recessive retinal degeneration (Human Gene Mutation Database). Nonsense mutations in RPGRIP1 have been reported in the majority of LCA6 patients (Won et al. 2009). Also, compromised interaction of RPGRIP1 with other proteins may contribute to the pathogenesis of primary open angle glaucoma (Fernández-Martínez et al. 2011).

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

All patients with symptoms suggestive of LCA, CRD and RP, especially patients with defects in disc morphogenesis.

Gene

Official Gene Symbol OMIM ID
RPGRIP1 605446
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Autosomal Recessive Retinitis Pigmentosa Sequencing Panel
Cone-Rod Dystrophy Sequencing Panel
Leber Congenital Amaurosis Sequencing Panel
Retinitis Pigmentosa (includes RPGR ORF15) Sequencing Panel
Stargardt Disease (STGD) and Macular Dystrophies (includes RPGR ORF15) Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Booij JC. et al. 2005. Journal of medical genetics. 42: e67. PubMed ID: 16272259
  • Chen Y, Zhang Q, Shen T, Xiao X, Li S, Guan L, Zhang J, Zhu Z, Yin Y, Wang P, Guo X, Wang J, et al. 2013. Comprehensive Mutation Analysis by Whole-Exome Sequencing in 41 Chinese Families With Leber Congenital Amaurosis. Investigative Ophthalmology & Visual Science 54: 4351–4357. PubMed ID: 23661368
  • Coene KLM, Mans DA, Boldt K, Gloeckner CJ, Reeuwijk J van, Bolat E, Roosing S, Letteboer SJF, Peters TA, Cremers FPM, Ueffing M, Roepman R. 2011. The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase. Human Molecular Genetics 20: 3592–3605. PubMed ID: 21685204
  • Cremers FP, Hurk JA van den, Hollander AI den. 2002. Molecular genetics of Leber congenital amaurosis. Human molecular genetics 11: 1169–1176. PubMed ID: 12015276
  • den Hollander AI, Roepman R, Koenekoop RK, Cremers FPM. 2008. Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog Retin Eye Res 27: 391–419. PubMed ID: 18632300
  • Fazzi E, Signorini SG, Scelsa B, Bova SM, Lanzi G. 2003. Leber’s congenital amaurosis: an update. Eur. J. Paediatr. Neurol. 7: 13–22. PubMed ID: 12615170
  • Fernández-Martínez L, Letteboer S, Mardin CY, Weisschuh N, Gramer E, Weber BH, Rautenstrauss B, Ferreira PA, Kruse FE, Reis A. 2011. Evidence for RPGRIP1 gene as risk factor for primary open angle glaucoma. European Journal of Human Genetics 19: 445–451. PubMed ID: 21224891
  • Human Gene Mutation Database (Bio-base).
  • Koenekoop RK. 2005. RPGRIP1 is mutated in Leber congenital amaurosis: a mini-review. Ophthalmic Genet. 26: 175–179. PubMed ID: 16352478
  • Li L, Xiao X, Li S, Jia X, Wang P, Guo X, Jiao X, Zhang Q, Hejtmancik JF. 2011. Detection of Variants in 15 Genes in 87 Unrelated Chinese Patients with Leber Congenital Amaurosis. PLoS ONE 6: e19458. PubMed ID: 21602930
  • Perrault I, Rozet JM, Calvas P, Gerber S, Camuzat A, Dollfus H, Châtelin S, Souied E, Ghazi I, Leowski C, Bonnemaison M, Paslier D Le, et al. 1996. Retinal-specific guanylate cyclase gene mutations in Leber’s congenital amaurosis. Nat. Genet. 14: 461–464. PubMed ID: 8944027
  • Roepman R, Letteboer SJ, Arts HH, Beersum SE van, Lu X, Krieger E, Ferreira PA, Cremers FP. 2005. Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosis-associated mutations. Proceedings of the National Academy of Sciences of the United States of America 102: 18520–18525. PubMed ID: 16339905
  • Weleber RG, Francis PJ, Trzupek KM, Beattie C. 2013. Leber Congenital Amaurosis. 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: 20301475
  • Weleber RG. 2002. Infantile and childhood retinal blindness: a molecular perspective (The Franceschetti Lecture). Ophthalmic Genet. 23: 71–97. PubMed ID: 12187427
  • Won J, Gifford E, Smith RS, Yi H, Ferreira PA, Hicks WL, Li T, Naggert JK, Nishina PM. 2009. RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis. Human Molecular Genetics 18: 4329–4339. PubMed ID: 19679561
  • Zhao Y, Hong D-H, Pawlyk B, Yue G, Adamian M, Grynberg M, Godzik A, Li T. 2003. The retinitis pigmentosa GTPase regulator (RPGR)-interacting protein: subserving RPGR function and participating in disk morphogenesis. Proceedings of the National Academy of Sciences 100: 3965–3970. PubMed ID: 12651948
Order Kits
TEST METHODS

NextGen Sequencing using PG-Designed 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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