Cataract 10, Multiple Types (CTRCT10) via the CRYBA1 Gene
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
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The great majority of tests are completed within 20 days.
The clinical sensitivity of this test may range up to 8%. In India, none of the 100 congenital cataract cases showed causative CRYBA1 sequence variants (Kumar et al. 2013). In Australia, 2.6% (1/38) of families with congenital cataract tested positive for disease-causing CRYBA1 sequence variants (Burdon et al. 2004). In China, 8% (2/25) of families with congenital cataracts harbored pathogenic sequence variants in the CRYBA1 gene (Sun et al. 2011).
Cataract 10, multiple types (CTRCT10) is a common, congenital, progressive, bilateral, symmetric, zonular, sutural vision disorder that causes blindness in infants (Hejtmancik 2008). It is characterized by the development of blurred and dimmed vision resulting from clouding of the lens (opacification) due to changes in its microarchitecture (Kumar et al. 2013). This particular damage to the lens induces light to scatter as well as proteins to aggregate, thereby resulting in loss of transparency (Hejtmancik 2008; Kumar et al. 2013). Fibers internal and external to the zonular cataract remain clear, and the position and thickness of the opaque layer varies from person to person, ranging from dense to dotted or dusty appearance (Padma et al. 1995). The incidence of congenital cataract has been estimated to be roughly 2.5 per 10,000 live births (Wirth et al. 2002; Yi et al. 2011). Perinatal ocular examination in newborns via red reflex examination is generally conducted using an ophthalmoscope (American Academy of Pediatrics 2002), whereas young children are assessed by slit-lamp microscopy (Li et al. 2013). Congenital cataract is usually treated by surgery and early primary intraocular lens implantation during the first year of life (Ventura et al. 2013).
CTRCT10 is an autosomal dominant vision disorder that is caused by pathogenic sequence variants in the crystallin, beta-A1 (CRYBA1) gene [also known as the crystallin, beta-1 (CRYB1) gene], which is located on chromosome 17q11.2 (Padma et al. 1995). The CRYBA1 gene consists of six coding exons, spanning approximately 8 kb, and encodes two proteins, namely, beta-A3- and beta-A1-crystallin. The production of two beta-crystallin proteins from a single CRYBA1 gene is attributable to the two potential initiation codons that are located in the first and second exons, with the shorter beta-A1 polypeptide differing by 17 amino acid residues at its N-terminus (Hogg et al. 1986; Werten et al. 1996; Lampi et al. 1997). Different beta-crystallin proteins are essential in the embryonic development of the lens and retina; these interact with each other and form oligomers of variable size (range: 2-8 units), as well as with other lens proteins to create a structural lattice. These protein-protein interactions also play a key role in maintaining lens transparency. To date, a total of about 8 pathogenic CRYBA1 sequence variants have been reported. These variants are mostly chain termination (splicing, deletion, frameshift) and a few missense variants (Human Gene Mutation Database). In vitro studies have suggested that these chain termination variants impair protein folding and solubility of beta-crystallin proteins (Reddy et al. 2004; Sinha et al. 2012).
For this NextGen test, the full coding regions plus ~10 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
The ideal CRYBA1 test candidates are individuals who present with congenital, autosomal dominant cataract.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - email@example.com
- Madhulatha Pantrangi, PhD - firstname.lastname@example.org
- American Academy of Pediatrics. 2002. Pediatrics. 109: 980-1. PubMed ID: 11986467
- Burdon K.P. et al. 2004. The British Journal of Ophthalmology. 88: 79-83. PubMed ID: 14693780
- Hejtmancik J.F. 2008. Seminars in cell & developmental biology. 19: 134-49. PubMed ID: 18035564
- Hogg D. et al. 1986. The Journal of Biological Chemistry. 261: 12420-7. PubMed ID: 3745196
- Human Gene Mutation Database (HGMD).
- Kumar M. et al. 2013. Molecular Vision. 19: 2436-50. PubMed ID: 24319337
- Lampi K.J. et al. 1997. The Journal of Biological Chemistry. 272: 2268-75. PubMed ID: 8999933
- Li LH. et al. 2013. The British Journal of Ophthalmology. 97: 588-91. PubMed ID: 23426739
- Padma T. et al. 1995. American Journal of Human Genetics. 57: 840-5. PubMed ID: 7573044
- Reddy M.A. et al. 2004. Human Molecular Genetics. 13: 945-53. PubMed ID: 15016766
- Sinha D. et al. 2012. Transgenic Research. 21: 1033-42. PubMed ID: 22427112
- Sun W. et al. 2011. Molecular vision. 17: 2197-206. PubMed ID: 21866213
- Ventura M.C. et al. 2013. Arquivos Brasileiros De Oftalmologia. 76: 240-3. PubMed ID: 24061837
- Werten P.J. et al. 1996. Protein Engineering. 9: 1021-8. PubMed ID: 8961355
- Wirth M.G. et al. 2002. The British Journal of Ophthalmology. 86: 782-6. PubMed ID: 12084750
- Yi J. et al. 2011. International Journal of Ophthalmology. 4: 422-32. PubMed ID: 22553694
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 ~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.
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 ~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.
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.