Leber Congenital Amaurosis 10 (LCA10) via CEP290 Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
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.
A study identified the c.2991+1655A>G CEP290 mutation in 21% (16/76 of LCA patients; 62.5% of all mutant alleles) of unrelated patients with LCA, either homozygously or compound heterozygously and reported it as the major LCA-associated gene of European ancestry (den Hollander et al. Am J Hum Genet 79(3): 556-561, 2006). The Perrault et al (2007) study also reported the high frequency of CEP290 mutations in their LCA cohort (22%; 21/96) and suggested that CEP290 is involved in families of European descent only (38/38) (Perrault et al. Hum Mutat 28(4):416, 2007). However, they reported 23 novel mutations and only 43% (33/76) of mutant alleles had c.2991+1655A>G mutations. Moreover, 12 families did not carry this common intronic causative mutation.
To date, no gross deletions have been reported in CEP290 that are associated with Leber congenital amaurosis 10 (The Human Gene Mutation Database).
Leber congenital amaurosis (LCA, OMIM# 204000) is the most severe form of inherited retinal dystrophy that is usually evident at birth or during the first months of life. LCA is clinically characterized by poor visual function often accompanied by sensory nystagmus, abnormal pupillary responses, high hyperopia, severely reduced visual acuity, photo-aversion, 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. GeneReviews, 2013). The estimated prevalence of LCA is 2-3 per 100,000 live births and accounts for 10-18% of congenital blindness (Fazzi et al. Eur J Paediatr Neurol 7(1):13-22, 2003).
LCA is a genetically heterogeneous disorder and is often inherited in an autosomal recessive manner. To date, approximately 19 genes have been implicated in the pathogenesis of different types of LCA (Weleber et al., 2013; Chen et al. Invest Ophthalmol Vis Sci 54(6):4351-4357, 2013). Together these genes account for 70% of the 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. Prog Retin Eye Res 27(4):391-419, 2008). Mutations in these genes cause not only LCA but also other retinal disorders (Weleber. Ophthalmic Genet 23(2):71-97, 2002). To date, mutations in CEP290 (also known as NPHP6, OMIM 610142) are the most frequent cause of LCA and are referred to as LCA10 (OMIM 611755) (den Hollander et al. Am J Hum Genet 79(3):556-561, 2006). CEP290, which is located on Chromosome 12, encodes a centrosomal protein that localizes primarily to centrosomes of dividing cells and to the connecting ciliated cells of inner and outer segments of the retinal photoreceptors (Chang et al. Hum Mol Genet 15(11):1847-1857, 2006). Perturbations in the photoreceptor ciliary transports due to mutations in CEP290 are associated with retinal degeneration as well as more pleiotropic phenotypes that include Joubert syndrome, Bardet-Biedl syndrome, lethal Meckel-Grüber syndrome (MKS), etc. (Adams et al. Ophthalmic Genet 28:113-125, 2007; Coppieters et al. Hum Mutat 31(10):E1709-1766, 2010; Yzer et al. Molecular Vision 18:412-425, 2012). Over 70 mutations in CEP290 account for ~21% of the LCA cases and two-thirds of all mutant alleles carry the intronic mutation c.2991+1655A>G. This mutation creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon (p.Cys998X). Genes with this mutation appear to produce a small amount of residual protein, which might be sufficient for normal cerebellar and renal function but not for the proper function of the photoreceptors and therefore leads specifically to the retinal degeneration (den Hollander et al., 2006).
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 test provides full coverage of all coding exons of the CEP290 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Candidates for this test are all LCA patients, patients with symptoms consistent with LCA10, family members of patients who have known mutations and carrier testing for at-risk family members.
|Official Gene Symbol||OMIM ID|
|Bardet-Biedl Syndrome 1||AR||209900|
|Joubert Syndrome 5||AR||610188|
|Leber Congenital Amaurosis 10||AR||611755|
|Meckel Syndrome 4||AR||611134|
|Senior-Loken Syndrome 6||AR||610189|
- Genetic Counselor Team - firstname.lastname@example.org
- Madhulatha Pantrangi, PhD - email@example.com
- Adams, N.A. et al. (2007). PubMed ID: 17896309
- Chang, B. et al. (2006). PubMed ID: 16632484
- Chen, Y. et al. (2013). PubMed ID: 23661368
- Coppieters, F. et al. (2010). PubMed ID: 20683928
- 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
- den Hollander, A.I. et al. (2006). PubMed ID: 16909394
- 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
- Human Gene Mutation Database (Bio-base).
- Perrault, I. et.al. (2007). PubMed ID: 17345604
- 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
- Yzer, S. et al. (2012). PubMed ID: 22355252
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.