Cataract 17, Multiple Types (CTRCT17) via the CRYBB1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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The great majority of tests are completed within 18 days.
The clinical sensitivity of this test may range up to 18%. In China, none of the 25 families with congenital cataracts showed pathogenic sequence variants in the CRYBB1 gene (Sun et al. 2011). In two independent studies in India, none of the 60 south Indian families with inherited pediatric cataract (Devi et al. 2008) and 8% (8/100) of congenital cataract cases (Kumar et al. 2013) showed disease-causing CRYBB1 sequence variants. In the United States, 4.3% (1/23) of families with autosomal dominant cataract tested positive for causative CRYBB1 sequence variants (Reis et al. 2013). In Saudi Arabia, 18% (7/38) of pediatric cataract patients harbored pathogenic sequence variants in the CRYBB1 gene (Aldahmesh et al. 2012).
Cataract 17 (CTRCT17) is a common congenital, bilateral, progressive, nuclear, pulverulent, sutural vision disorder that causes blindness in children (Mackay et al. 2002). 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). Intrafamilial variability in the morphology and location within the lens commonly occurs in CTRCT17 (Yang et al. 2008). The incidence of congenital cataract has been estimated to be roughly 2 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). CTRCT17 has been strongly associated with microcornea (Willoughby et al. 2005).
CTRCT17 is an autosomal dominant vision disorder that is caused by pathogenic sequence variants in the crystallin, beta-B1 (CRYBB1) gene, which is located on chromosome 22q12.1 (Mackay et al. 2002). The CRYBB1 gene consists of five coding exons that encode a 252-amino acid structural protein called beta-crystallin B1, which is expressed in the lens tissue and plays an important structural role in the maintenance of lens transparency and refractive index (David et al. 1996). The CRYBB1 protein has a significantly longer N-terminal extension compared to the other two beta-crystallins, namely CRYBB2 and CRYBB3, thereby allowing the formation of higher molecular weight protein aggregates (Den Dunnen et al. 1986; Ajaz et al. 1997). Pathogenic sequence variants in the CRYBB1 gene significantly reduce the stability of the beta-crystallin B1 monomer via deamidation, thus disrupting the formation of hetero-oligomers and protein folding, which are critical for lens transparency (Harms et al. 2004; Wang et al. 2010; Wang et al. 2013). To date, a total of over 10 pathogenic CRYBB1 sequence variants have been reported, which are mostly missense and a few chain terminations (nonsense and frame shift) (Human Gene Mutation Database). The autosomal recessive form of CTRCT17 less frequency occurs and involves the abrogation of the N terminal region, thus leading to nonsense-mediated decay and ultimately no protein product (Cohen et al. 2007; Meyer et al. 2009).
This test involves bidirectional DNA Sanger sequencing of all coding exons of the CRYBB1 gene. The full coding region of each exon plus ~10 bp of flanking non-coding DNA on either side are sequenced. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.
Indications for Test
The ideal CRYBB1 test candidates are individuals who present with congenital, bilateral, progressive, nuclear, pulverulent, sutural autosomal dominant cataract.
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|Congenital Cataracts Sequencing Panel|
- Genetic Counselor Team - email@example.com
- Madhulatha Pantrangi, PhD - firstname.lastname@example.org
- Ajaz M.S. et al. 1997. The Journal of Biological Chemistry. 272: 11250-5. PubMed ID: 9111027
- Aldahmesh M.A. et al. 2012. Genetics in Medicine. 14: 955-62. PubMed ID: 22935719
- American Academy of Pediatrics. 2002. Pediatrics. 109: 980-1. PubMed ID: 11986467
- Cohen D. et al. 2007. Investigative Ophthalmology & Visual Science. 48: 2208-2213. PubMed ID: 17460281
- David L.L. et al. 1996. The Journal of Biological Chemistry. 271: 4273-9. PubMed ID: 8626774
- den Dunnen J.T. et al. 1986. Proceedings of the National Academy of Sciences of the United States of America. 83: 2855-9. PubMed ID: 3458246
- Devi R.R. et al. 2008. Molecular Vision. 14: 1157-70. PubMed ID: 18587492
- Harms MJ. et al. 2004. Protein Science : a Publication of the Protein Society. 13: 678-86. PubMed ID: 14978307
- Hejtmancik J.F. 2008. Seminars in cell & developmental biology. 19: 134-49. PubMed ID: 18035564
- Human Gene Mutation Database (Bio-base).
- Kumar M. et al. 2013. Molecular Vision. 19: 2436-50. PubMed ID: 24319337
- Li L.H. et al. 2013. The British Journal of Ophthalmology. 97: 588-91. PubMed ID: 23426739
- Mackay D.S. et al. 2002. American Journal of Human Genetics. 71: 1216-21. PubMed ID: 12360425
- Meyer E. et al. 2009. Molecular Vision. 15: 1014-9. PubMed ID: 19461930
- Reis L.M. et al. 2013. Human genetics. 132: 761-70. PubMed ID: 23508780
- 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
- Wang K.J. et al. 2011. Human Mutation. 32: E2050-60. PubMed ID: 21972112
- Wang S. et al. 2013. Biochimica Et Biophysica Acta. 1832: 302-11. PubMed ID: 23159606
- Willoughby C.E. et al. 2005. Molecular Vision. 11: 587-93. PubMed ID: 16110300
- Wirth M.G. et al. 2002. The British Journal of Ophthalmology. 86: 782-6. PubMed ID: 12084750
- Yang J. et al. 2008. Molecular Vision. 14: N/A. PubMed ID: 18432316
- Yi J. et al. 2011. International Journal of Ophthalmology. 4: 422-32. PubMed ID: 22553694
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.
As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all 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 for example 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 and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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.