Cystic Fibrosis and CF-Related Disorders via CFTR 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.
CPT 81220 can be used for CFTR common variant analysis.
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.
Our full gene sequencing test is expected to detect >98% of CFTR causative mutations involved in Cystic Fibrosis or CF-related disorders like CBAVD and pancreatitis.
To date, no gross duplications or deletions have been reported for CFTR-associated pancreatitis and a relatively small number of large deletions and other major gene rearrangements have been reported to cause cystic fibrosis and CBAVD (Human Gene Mutation Database).
Cystic Fibrosis (CF) is a severe disorder with typical onset in infancy. Main features include chronic, debilitating lung infections, pancreatic insufficiency connected to dietary malabsorption; excess Cl- in sweat, and absence of the vas deferens. Severity varies widely; genetic and non-genetic modifying factors exist. Average survival in patients is currently ~37 years. Today, many U.S. CF cases are detected through routine neonatal screening (http://genes-r-us.uthscsa.edu/). CF is one of the most common “single gene” disorders with carrier frequencies up to 1/25 in individuals with Northern European ancestry.
CF-Related disorders include chronic pancreatitis due to pancreatic insufficiency and congenital bilateral absence of the vas deferens (CBAVD) in the absence of other CF features (Lebo and Grody, 2007). In the case of CBAVD, nearly all male patients are infertile.
Cystic Fibrosis is caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene. Inheritance is autosomal recessive. Over 1300 causative CFTR mutations have been reported; most are very rare (http://genes-r-us.uthscsa.edu/). Missense mutations predominate, but there are also substantial numbers of nonsense, frameshift, and splicing mutations. A relatively small number of larger deletions and other major gene rearrangements have been reported. One major mutation, ΔF508, comprises about 70% of all mutant alleles in Northern Europeans. Correlations between genotyping and phenotype are beginning to be made (Moskowitz et al. 2005; McKone et al. 2006).
The CFTR gene encodes a membrane channel protein which transports chloride ions across cells that produce mucus, saliva, sweat, tears and digestive enzymes (Kreda, S. et al., 2012). CFTR also regulates the function of sodium ion channels, which is necessary for the function of the lungs and pancreas. Though CFTR mutations are typically associated with cystic fibrosis, and CBAVD, recent literature has demonstrated the presence of CFTR variants in individuals with chronic pancreatitis (Masson, E. et al., 2013).
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 CFTR gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
This test also includes sequencing of regions within intron 11 and intron 21, along with a complete analysis of the compound (TG)n(T)n sequence (5T/TG tract) in intron 8. Variations in the 5T/TG tract have been reported to cause CBAVD (Chillon et al. 1995). Our 5T/TG tract analysis involves bidirectional sequencing along with allele length measurement.
Indications for Test
Most CF patients and many potential CFTR mutation carriers will first receive testing for a collection of ≥23 specific, well characterized mutations (Amos et al., 2011). PreventionGenetics does not offer such a panel at this time. Patients, who carry no more than one of the mutations within the panel, are candidates for our full gene sequencing test. CF patients with Native American, African or Asian ancestry may also be candidates for the full sequencing test, as the standard panel of mutations was designed for Caucasian patients. CBAVD patients are also good candidates for our full sequencing test, especially since the 5T/TG tract has a particularly strong effect on this phenotype. The 5T/TG tract analysis may be ordered alone. To rule out CF related pancreatitis, individuals with the following criteria should consider testing for mutations in CFTR (Ellis et al., 2001):
Recurrent unexplained attacks of acute pancreatitis and a positive family history Unexplained chronic pancreatitis and a positive family history Unexplained chronic pancreatitis without a positive family history after exclusion of other causes such as hyperlipidaemia type I, familiar hypercalciuric hypercalcemia (FBH), hereditary hyperthyroidism and autoimmune pancreatitis. Unexplained pancreatitis episodes in children Individuals who test negative for mutations in the PRSS1 gene or have a single, heterozygous mutation in either SPINK1 or CTRC.
|Official Gene Symbol||OMIM ID|
|Congenital Bilateral Absence Of The Vas Deferens||AR||277180|
- Genetic Counselor Team - firstname.lastname@example.org
- Luke Drury, PhD - email@example.com
- Amos, J, Feldman, GL, Grody, WW, Monaghan, K, Palomaki, GE, Prior, TW, Richards, CS, Watson, MS. 20011. Technical Standards and Guidelines for CFTR Mutation Testing.
- Chillon, M, Casals, T, Mercier, B, Bassas, L, Lissens, W, Silber, S, Romey, MC, Ruiz-Romero, J, Verlingue, C, Claustres, M, et al. 1995. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N. Engl. J. Med. 332: 1475-1480. PubMed ID: 7739684
- Ellis, I, Lerch, MM, Whitcomb, DC. 2001. Genetic testing for hereditary pancreatitis: guidelines for indications, counselling, consent and privacy issues. Pancreatology 1:405-415. PubMed ID: 12120217
- Human Gene Mutation Database (Bio-base).
- Joergensen, MT, Brusgaard, K, Crüger, DG, Gerdes, AM, Schaffalitzky de Muckadell, OB. 2010. Genetic, epidemiological, and clinical aspects of hereditary pancreatitis: a population-based cohort study in Denmark. Am. J. Gastroenterol. 105:1876-1883. PubMed ID: 20502448
- Kreda, SM, Davis, CW, Rose, MC. 2012. CFTR, mucins, and mucus obstruction in cystic fibrosis. Cold Spring Harb. Perspect. Med. 2:a009589. PubMed ID: 22951447
- Lebo, RV, Grody, WW. 2007. Variable penetrance and expressivity of the splice altering 5T sequence in the cystic fibrosis gene. Genet. Test 11: 32-44. PubMed ID: 17394391
- Masson, E, Chen, JM, Audrézet, MP, Cooper, DN, Férec, C. 2013. A Conservative Assessment of the Major Genetic Causes of Idiopathic Chronic Pancreatitis: Data from a Comprehensive Analysis of PRSS1, SPINK1, CTRC and CFTR Genes in 253 Young French Patients. PLoS One 8:e73522. PubMed ID: 23951356
- McKone, EF, Goss, CH, Aitken, ML. 2006. CFTR genotype as a predictor of prognosis in cystic fibrosis. Chest 130: 1441-1447. PubMed ID: 17099022
- Moskowitz, SM, Chmiel, JF, Sternen, DL, Cheng, E, Cutting, GR. 2008. CFTR-Related Disorders. GeneReviews. PubMed ID: 20301428
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.