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Bleeding Disorders Sequencing Panel
- Summary and Pricing
- Clinical Features and Genetics
- Citations
- Methods
- Ordering/Specimens
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TEST METHODS
- NextGen Sequencing using PG-Select Capture Probes
- Deletion/Duplication Testing via Array Comparative Genomic Hybridization
NGS Sequencing
| Test Code | Test Copy Genes | CPT Code Copy CPT Codes | |
|---|---|---|---|
| 1375 | ABCG5 | 81479 | Add |
| ABCG8 | 81479 | ||
| ACTN1 | 81479 | ||
| ADAMTS13 | 81479 | ||
| ANKRD26 | 81479 | ||
| ANO6 | 81479 | ||
| AP3B1 | 81479 | ||
| BLOC1S3 | 81479 | ||
| CD36 | 81479 | ||
| CYCS | 81479 | ||
| DTNBP1 | 81479 | ||
| F10 | 81479 | ||
| F11 | 81479 | ||
| F12 | 81479 | ||
| F13A1 | 81479 | ||
| F13B | 81479 | ||
| F2 | 81479 | ||
| F5 | 81479 | ||
| F7 | 81479 | ||
| F8 | 81407 | ||
| F9 | 81238 | ||
| FGA | 81479 | ||
| FGB | 81479 | ||
| FGG | 81479 | ||
| FLI1 | 81479 | ||
| FLNA | 81479 | ||
| GATA1 | 81479 | ||
| GFI1B | 81479 | ||
| GGCX | 81479 | ||
| GP1BA | 81479 | ||
| GP1BB | 81404 | ||
| GP6 | 81479 | ||
| GP9 | 81479 | ||
| HOXA11 | 81479 | ||
| HPS1 | 81479 | ||
| HPS3 | 81479 | ||
| HPS4 | 81479 | ||
| HPS5 | 81479 | ||
| HPS6 | 81479 | ||
| ITGA2 | 81479 | ||
| ITGA2B | 81479 | ||
| ITGB3 | 81479 | ||
| LMAN1 | 81479 | ||
| MASTL | 81479 | ||
| MCFD2 | 81479 | ||
| MPL | 81479 | ||
| MYH9 | 81479 | ||
| NBEAL2 | 81479 | ||
| P2RX1 | 81479 | ||
| P2RY12 | 81479 | ||
| PLAU | 81479 | ||
| PRKACG | 81479 | ||
| RUNX1 | 81479 | ||
| SERPINE1 | 81479 | ||
| SERPINF2 | 81479 | ||
| TBXA2R | 81479 | ||
| TBXAS1 | 81479 | ||
| TUBB1 | 81479 | ||
| VKORC1 | 81479 | ||
| VWF | 81408 | ||
| WAS | 81406 | ||
| Full Panel Price* | $790.00 | ||
| Test Code | Test Copy Genes | Total Price | CPT Codes Copy CPT Codes | |
|---|---|---|---|---|
| 1375 | Genes x (61) |
$790.00 | 81238, 81404, 81406, 81407, 81408, 81479(x56) | Add |
Pricing Comment
We are happy to accommodate requests for single genes 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 on our PGxome Custom Panel.
Targeted Testing
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
Turnaround Time
The great majority of tests are completed within 20 days.
Clinical Sensitivity
This test is designed to identify variants in genes associated with a variety of bleeding phenotypes. In a recent study of patients with excessive bleeding, a next generation sequencing approach was used for differential diagnosis. Of the 61 patients with a suspected etiology, a corresponding pathogenic variant was identified in 91.8% of cases. In 76 patients with an unknown etiology, a causative pathogenic variant was identified in 10.5% of cases (Simeoni et al. 2016).
Deletion/Duplication Testing via aCGH
| Test Code | Test Copy Genes | Individual Gene Price | CPT Code Copy CPT Codes | |
|---|---|---|---|---|
| 600 | ADAMTS13 | $990.00 | 81479 | Add |
| ANKRD26 | $990.00 | 81479 | ||
| ANO6 | $990.00 | 81479 | ||
| AP3B1 | $990.00 | 81479 | ||
| BLOC1S3 | $990.00 | 81479 | ||
| CD36 | $990.00 | 81479 | ||
| DTNBP1 | $990.00 | 81479 | ||
| F10 | $990.00 | 81479 | ||
| F11 | $990.00 | 81479 | ||
| F12 | $990.00 | 81479 | ||
| F13A1 | $990.00 | 81479 | ||
| F13B | $990.00 | 81479 | ||
| F2 | $990.00 | 81479 | ||
| F5 | $990.00 | 81479 | ||
| F7 | $990.00 | 81479 | ||
| F8 | $990.00 | 81406 | ||
| F9 | $990.00 | 81479 | ||
| FGA | $990.00 | 81479 | ||
| FGB | $990.00 | 81479 | ||
| FGG | $990.00 | 81479 | ||
| FLNA | $990.00 | 81479 | ||
| GATA1 | $990.00 | 81479 | ||
| GGCX | $990.00 | 81479 | ||
| GP1BA | $990.00 | 81479 | ||
| GP1BB | $990.00 | 81479 | ||
| GP6 | $990.00 | 81479 | ||
| GP9 | $990.00 | 81479 | ||
| HPS1 | $990.00 | 81479 | ||
| HPS3 | $990.00 | 81479 | ||
| HPS4 | $990.00 | 81479 | ||
| HPS5 | $990.00 | 81479 | ||
| HPS6 | $990.00 | 81479 | ||
| ITGA2 | $990.00 | 81479 | ||
| ITGA2B | $990.00 | 81479 | ||
| ITGB3 | $990.00 | 81479 | ||
| LMAN1 | $990.00 | 81479 | ||
| MASTL | $990.00 | 81479 | ||
| MCFD2 | $990.00 | 81479 | ||
| MPL | $990.00 | 81479 | ||
| MYH9 | $990.00 | 81479 | ||
| NBEAL2 | $990.00 | 81479 | ||
| P2RX1 | $990.00 | 81479 | ||
| P2RY12 | $990.00 | 81479 | ||
| PLAU | $990.00 | 81479 | ||
| RUNX1 | $990.00 | 81479 | ||
| SERPINE1 | $990.00 | 81479 | ||
| SERPINF2 | $990.00 | 81479 | ||
| TBXA2R | $990.00 | 81479 | ||
| TBXAS1 | $990.00 | 81479 | ||
| VKORC1 | $990.00 | 81479 | ||
| VWF | $990.00 | 81479 | ||
| WAS | $990.00 | 81479 | ||
| Full Panel Price* | $1490.00 | |||
| Test Code | Test Copy Genes | Total Price | CPT Codes Copy CPT Codes | |
|---|---|---|---|---|
| 600 | Genes x (52) |
$1490.00 | 81406, 81479(x51) | Add |
Pricing Comment
|
# of Genes Ordered |
Total Price |
|
1 |
$990 |
|
2-5 |
$1190 |
|
6-10 |
$1290 |
|
11-100 |
$1490 |
|
Over 100 |
Call for quote |
Turnaround Time
The great majority of tests are completed within 20 days.
Clinical Sensitivity
Large deletions in various inherited platelet disorders represent ~20% of causative variants found within AP3B1 and HPS3, ~10% in HPS6 and GP1BB, 2% in GP1BA, 5% in ITGA2B, 2% in ITGB3, and 100% in PLAU (Human Gene Mutation Database).
For coagulation deficiency genes, large deletions are represent 5% of causative variants in the F10, 2% in F11, 3% in F13A1, 2% in F5, 2% in F7, 6% in F8, 3% in F9, 15% in FGA, and 5% in VWF (Human Gene Mutation Database).
The majority of variants reported in the thrombocytopenia genes in this panel are missense and nonsense variants. Large deletions account for ~24% of the reported RUNX1 gene variants, but in general, large, multi-exon and whole gene deletions are rare among the thrombocytopenia panel genes. In addition to the RUNX1 gene, large deletions have also been reported in the GP1BB, MYH9, and WAS genes (Human Gene Mutation Database).
Clinical Features
This sequencing panel focuses on inherited bleeding disorders due to impaired platelet function, coagulation factor deficiencies or thrombocytopenia. With many factors contributing to clot formation, differential diagnosis of the various bleeding disorders can be time-intensive, labor-intensive, and difficult to interpret, especially in patients with milder symptoms. Genetic testing provides a means to examine multiple bleeding disorder genes simultaneously to quickly identify potential causes to disease. Differential diagnosis is especially helpful in employing appropriate therapies to mitigate bleeding episodes (Westbury et al. 2015; Simeoni et al. 2016; Lentaigne et al. 2016).
Platelet function disorders (PFDs) tested in this panel include defects in platelet adhesion/coagulation, receptor function, or secretion (Handin et al 2005). PFDs due to defects in platelet adhesion include Bernard-Soulier syndrome and Scott Syndrome; defects in receptor function include Glanzmann’s thrombasthenia, Thromboxane A2 receptor deficiency, GPVI collagen receptor deficiency, and P2Y12 ADP receptor deficiency. Defects in storage granules include Hermansky-Pudlak Syndrome.
Coagulation factor deficiency panel includes testing for a large group of inherited bleeding disorders including three types of hemophilia, von Willebrand disease and rare bleeding disorders (RBD). RBDs include inherited deficiencies in fibrinogen, factor (F) II, FV, FV +FVIII, FVII, FX, FXI, FXIII, plasminogen activator inhibitor, and alpha-s-plasmin inhibitor.
Inherited thrombocytopenias comprise a heterogeneous group of rare disorders characterized by low platelet counts. In adults, low platelet numbers are typically considered below 150,000/microL. Bleeding manifestations of thrombocytopenia include primarily excessive bruising (purpura), petechiae, prolonged bleeding from cuts or from surgical procedures, spontaneous nose bleeds, and in women, heavy menstrual flows. Thrombocytopenia and consequent bleeding diatheses range in severity from mild to severe. About half of the inherited thrombocytopenias are syndromic disorders characterized by physical and neurological anomalies, and immunodeficiencies (Balduini et al. 2013). Some inherited thrombocytopenias are associated with an increased risk of developing myelodysplastic syndrome (MDS) and acute leukemia (AL) (Churpek et al. 2013). It is important to distinguish inherited thrombocytopenias from immune / idiopathic thrombocytopenias (ITP) in order to inform clinical management and identify potential at risk family members.
Genetics
Inherited platelet function disorders are inherited in an autosomal recessive manner due to pathogenic variants in the ITGB3, ITGA2B, AP3B1, BLOCK1S3, DTNBP1, HPS1, HPS3, HPS4, HPS5, HPS6, ANO6, GP1BA, GP9, GP1BB, P2RY12, CD36 and GP6 genes (Handin et al. 2005; Watson et al. 2013). Autosomal dominant forms include pathogenic variants in the TBXA2R, PLAU, PTGS1, and TBXAS1 genes.
Hemophilia A and B are inherited through an X-linked recessive manner through pathogenic variants in the F8 and F9 genes respectively and primarily affect males. VWD is inherited in both autosomal dominant and recessive manners through pathogenic variants in the VWF gene. RBDs are all inherited in an autosomal recessive manner with deficiencies in FVII, FXI, or FV accounting for ~80% of cases. RBD genes include FGA, FGB, FGG, F2, F5, F7, F10, F11, F12, F13A1, F13B, MCFD2, LMAN1, SERPINE1, SERPINF2, VKORC1, and GGCX. See individual test descriptions for additional information on the molecular biology of each gene.
Thrombocytopenia genes included in this panel have been associated with both syndromic and non-syndromic forms of inherited thrombocytopenia and represent the most well-documented forms of inherited thrombocytopenia reported in the literature. Thrombocytopenias are typically divided into three distinct groups based upon platelet size: large / macrothrombocytopenias, small / microthrombocytopenias, and thrombocytopenias with normal sized platelets (Westbury et al. 2015; Simeoni et al. 2016; Lentaigne et al. 2016).
Testing Strategy
For this NGS test, the full coding regions plus ~10bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, 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, undocumented and questionable variant calls are confirmed by Sanger sequencing. This test does not currently detect inversions in the F8 gene which account for about 40% of Hemophilia A cases.
This panel provides 100% coverage of the aforementioned regions of the indicated genes. We define coverage as > 20X NGS reads for exons and 0-10 bases of flanking DNA, > 10X NGS reads for 11-20 bases of flanking DNA, or Sanger sequencing.
Indications for Test
Candidates for testing include patients with excessive bleeding of unknown etiology due to abnormal platelet count, volume, morphology, function, or impaired coagulation. This test especially aids in a rapid differential diagnosis of phenotypically similar disorders, rules out particular syndromes, and provides the analysis of multiple genes simultaneously. Individuals who are suspected of any of these disorders, especially if clinical diagnosis is unclear and individuals who have been found to be negative by mutation analysis for a single gene test are candidates (Othman 2013; Peyvandi et al. 2012).
Genes
| Inheritance | Abbreviation |
|---|---|
| Autosomal Dominant | AD |
| Autosomal Recessive | AR |
| X-Linked | XL |
| Mitochondrial | MT |
Diseases
Related Tests
CONTACTS
Genetic Counselors
- Genetic Counselor Team - clinicaldnatesting@preventiongenetics.com
Geneticist
- Michael Chicka, PhD - michael.chicka@preventiongenetics.com
Citations
- Balduini C.L. et al. 2013. Journal of Thrombosis and Haemostasis. 11: 1006-19. PubMed ID: 23510089
- Churpek J.E. et al. 2013. Leukemia & Lymphoma. 54: 28-35. PubMed ID: 22691122
- Handin R.I. 2005. Hematology. American Society of Hematology Education Program. 396-402. PubMed ID: 16304410
- Human Gene Mutation Database (Bio-base).
- Klopocki E. et al. 2006. European Journal of Human Genetics. 14: 1274-9. PubMed ID: 16896345
- Lentaigne C. et al. 2016. Blood. 127: 2814-23. PubMed ID: 27095789
- Othman M. 2013. Seminars in Thrombosis and Hemostasis. 39: 575-8. PubMed ID: 23982907
- Papoulidis .I et al. 2014. Molecular Medicine Reports. 9: 163-5. PubMed ID: 24220582
- Peyvandi F. et al. 2012. Haemophilia. 18 Suppl 4: 148-53. PubMed ID: 22726099
- Simeoni I. et al. 2016. Blood. 127: 2791-803. PubMed ID: 27084890
- Watson S.P. et al. 2013. Journal of Thrombosis and Haemostasis. 11 Suppl 1: 351-63. PubMed ID: 23516995
- Westbury S.K. et al. 2015. Genome Medicine. 7: 36. PubMed ID: 25949529
Order Kits
TEST METHODS
- NextGen Sequencing using PG-Select Capture Probes
- Deletion/Duplication Testing via Array Comparative Genomic Hybridization
NextGen Sequencing using PG-Select Capture Probes
Test Procedure
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.
Analytical Validity
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.
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 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.
Deletion/Duplication Testing via Array Comparative Genomic Hybridization
Test Procedure
Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.
Analytical Validity
PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.
Analytical Limitations
Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.
This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.
aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.
Breakpoints, if occurring outside the targeted gene, may be hard to define.
The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.
Order Kits
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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