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Primary Ciliary Dyskinesia (PCD)/Immotile Cilia Syndrome and Cystic Fibrosis Sequencing Panel with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing with CNV

Test Code Test Copy GenesCPT Code Copy CPT Codes
10415 AK7 81479,81479 Add to Order
ARMC4 81479,81479
CCDC103 81479,81479
CCDC114 81479,81479
CCDC151 81479,81479
CCDC39 81479,81479
CCDC40 81479,81479
CCDC65 81479,81479
CCNO 81479,81479
CFAP298 81479,81479
CFTR 81223,81222
DNAAF1 81479,81479
DNAAF2 81479,81479
DNAAF3 81479,81479
DNAAF4 81479,81479
DNAAF5 81479,81479
DNAH1 81479,81479
DNAH11 81479,81479
DNAH5 81479,81479
DNAH8 81479,81479
DNAI1 81479,81479
DNAI2 81479,81479
DNAJB13 81479,81479
DNAL1 81479,81479
DRC1 81479,81479
GAS8 81479,81479
INVS 81479,81479
LRRC6 81479,81479
MCIDAS 81479,81479
NME8 81479,81479
OFD1 81479,81479
PIH1D3 81479,81479
RPGR 81479,81479
RSPH1 81479,81479
RSPH3 81479,81479
RSPH4A 81479,81479
RSPH9 81479,81479
SPAG1 81479,81479
TTC25 81479,81479
ZMYND10 81479,81479
Full Panel Price* $930
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
10415 Genes x (40) $930 81222, 81223, 81479(x78) Add to Order

New York State Approved Test

Pricing Comments

We are happy to accommodate requests for testing single genes in this panel or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered via our PGxome Custom Panel tool.

Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

Clinical sensitivity for the Primary Ciliary Dyskinesia NGS test is ~80% (Zariwala et al. 2013).

Gross deletions or duplications not detectable by sequencing have been reported in ARMC4, CCDC40, CFTR, DNAAF1, DNAAF2, DNAH11, DNAH5, DNAAF4 (previously called DYX1C1), SPAG1, OFD1, PIH1D3, RPGR, and ZMYND10 (Human Gene Mutation Database). Clinical sensitivity is difficult to predict due to the paucity of documented cases, but is expected to be low.

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Clinical Features

Primary Ciliary Dyskinesia (PCD) is a genetic disorder affecting the function of motile cilia (Leigh et al. 2009). The hallmark features of PCD are neonatal respiratory distress, chronic coughing, and recurrent sinus and/or ear infections; 80-100% of all PCD patients have one or more of these symptoms. In 20-50% of individuals with PCD, the major visceral organs are reversed from their normal positions (situs inversus) (Sutherland and Ware 2009). Kartagener’s syndrome is a condition defined by the symptomatic triad of situs inversus, sinusitis and bronchiectasis. Patients with PCD can also have abnormal orientation of some organs but not others (a condition called situs ambiguus or heterotaxy) (Kennedy et al. 2007).

Genetics

Primary Ciliary Dyskinesia is caused by defects in motile cilia. Planar motion cilia (from the respiratory tract, brain, and reproductive tract) consist of nine microtubule doublets that surround a central core of two microtubules (9+2 configuration). Rotary motion cilia (those in the embryonal node) lack the central core microtubules (9+0 configuration). All motile cilia have inner and outer dynein arms attached at regular intervals to the nine peripheral microtubule doublets, which serve as molecular motors that drive microtubule sliding. For 9+2 cilia, radial spokes form a signal-transduction scaffold between the peripheral dynein arms and the central-core microtubule pair, giving these cilia their characteristic planar (forward and backward) motion.

Motile cilia are very complex structures composed of roughly 250 proteins (Ferkol and Leigh 2006). To date, defects in over 30 genes have been reported to cause PCD, which is most commonly inherited in an autosomal recessive manner (Zariwala et al. 2013). Rarely, PCD has been found to be inherited in an X-linked manner due to loss-of-function variants in OFD1, RPGR or PIH1D3 (Budny et al. 2006; Moore et al 2006; Olcese et al. 2017). In addition, the INVS/NPHP2 gene has been associated with situs inversus either with or without biliary complications (Schön et al. 2002; Otto et al. 2003). Symptoms of cystic fibrosis can sometimes mimic those of PCD. See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

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.

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 panel typically provides ≥98% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

This test is for patients with symptoms of respiratory distress (chronic coughing, recurrent sinusitis, bronchiectasis), with or without situs inversus. For patients with laterality defects (Heterotaxy, Situs Inversus, Situs ambiguous) but no overt symptoms of respiratory distress, see the Heterotaxy and Kartagener's Syndromes NextGen Panel (#1060).

Diseases

Name Inheritance OMIM ID
Ciliary Dyskinesia, Primary, 1 AR 244400
Ciliary Dyskinesia, Primary, 10 AR 612518
Ciliary Dyskinesia, Primary, 11 AR 612649
Ciliary Dyskinesia, Primary, 12 AR 612650
Ciliary Dyskinesia, Primary, 13 AR 613193
Ciliary Dyskinesia, Primary, 14 AR 613807
Ciliary Dyskinesia, Primary, 15 AR 613808
Ciliary Dyskinesia, Primary, 16 AR 614017
Ciliary Dyskinesia, Primary, 17 AR 614679
Ciliary Dyskinesia, Primary, 18 AR 614874
Ciliary Dyskinesia, Primary, 19 AR 614935
Ciliary Dyskinesia, Primary, 2 AR 606763
Ciliary Dyskinesia, Primary, 20 AR 615067
Ciliary Dyskinesia, Primary, 21 AR 615294
Ciliary Dyskinesia, Primary, 22 AR 615444
Ciliary Dyskinesia, Primary, 23 AR 615451
Ciliary Dyskinesia, Primary, 24 AR 615481
Ciliary Dyskinesia, Primary, 25 AR 615482
Ciliary Dyskinesia, Primary, 26 AR 615500
Ciliary Dyskinesia, Primary, 27 AR 615504
Ciliary Dyskinesia, Primary, 28 AR 615505
Ciliary Dyskinesia, primary, 29 AR 615872
Ciliary Dyskinesia, Primary, 3 AR 608644
Ciliary Dyskinesia, Primary, 30 AR 616037
Ciliary Dyskinesia, Primary, 32 AR 616481
Ciliary Dyskinesia, Primary, 33 AR 616726
Ciliary Dyskinesia, Primary, 34 AR 617091
Ciliary Dyskinesia, Primary, 35 AR 617092
Ciliary Dyskinesia, Primary, 36 XL 300991
Ciliary Dyskinesia, Primary, 37 AR 617577
Ciliary Dyskinesia, Primary, 6 AR 610852
Ciliary Dyskinesia, Primary, 7 AR 611884
Ciliary Dyskinesia, Primary, 9 AR 612444
Cystic Fibrosis AR 219700

Related Test

Name
PGxome®

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Budny B. et al. 2006. Human Genetics. 120: 171-8. PubMed ID: 16783569
  • Ferkol T., Leigh M. 2006. Seminars in Perinatology. 30: 335-40. PubMed ID: 17142159
  • Human Gene Mutation Database (Bio-base).
  • Kennedy M.P. et al. 2007. Circulation. 115: 2814-21. PubMed ID: 17515466
  • Leigh M.W. et al. 2009. Genetics in Medicine. 11: 473-87. PubMed ID: 19606528
  • Moore A. et al. 2006. Journal of Medical Genetics. 43: 326-33. PubMed ID: 16055928
  • Olcese C. et al. 2017. Nature Communications. 8: 14279. PubMed ID: 28176794
  • Otto EA. et al. 2003. Nature Genetics. 34: 413-20. PubMed ID: 12872123
  • Schön P. et al. 2002. Human Genetics. 110: 157-65. PubMed ID: 11935322
  • Sutherland M.J., Ware S.M. 2009. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. 151C: 307-17. PubMed ID: 19876930
  • Zariwala M.A. et al. 2013. Primary Ciliary Dyskinesia. 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: 20301301
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For PGxome® we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus 10 bases of flanking non-coding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

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.

Analytical Validity

NextGen Sequencing: 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.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

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 sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for all available transcripts plus 10 bp of flanking non-coding DNA for each exon. We also sequence other regions within or near genes in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere.  Unless specifically indicated, test reports contain no information about other portions of genes.

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 amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken 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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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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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