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Thrombocytopenia Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1951 ADAMTS13 81479 Add to Order
ANKRD26 81479
CYCS 81479
GATA1 81479
GP1BA 81479
GP1BB 81404
GP9 81479
MASTL 81479
MPL 81479
MYH9 81479
RUNX1 81479
WAS 81406
Full Panel Price* $1590.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1951 Genes x (12) $1590.00 81404, 81406, 81479(x10) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

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 28 days.

Clinical Sensitivity

This test is designed to identify variants in some of the more common genes associated with thrombocytopenias. While the overall clinical sensitivity of this panel is difficult to determine, in a recent study of 272 patients with macrothrombocytopenia, ~48% of patients harbored pathogenic variants in either the MYH9 gene (~ 38%), the Bernard Soulier genes (~ 10%), or the GATA1 gene (< 1%) (Kunishima, S. Thrombocytopenia, ISTH Webinar 2015: WEB150325). In general, ~ 50% of inherited thrombocytopenias are the result of disorders that are not yet characterized (Balduini et al. 2013).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ADAMTS13$690.00 81479 Add to Order
ANKRD26$690.00 81479
GATA1$690.00 81479
GP1BA$690.00 81479
GP1BB$690.00 81479
GP9$690.00 81479
MASTL$690.00 81479
MPL$690.00 81479
MYH9$690.00 81479
RUNX1$690.00 81479
WAS$690.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (11) $1290.00 81479(x11) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

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).

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Clinical Features

Thrombocytopenia is a blood disorder 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 with additional defects including 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

This panel test is designed to detect variants in several genes associated with either autosomal dominant, autosomal recessive, or X-linked forms of inherited thrombocytopenias. The genes included in this panel have been associated with both syndromic and non-syndromic forms of inherited thrombocytopenia and represent some of the more common 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.

Macrothrombocytopenias

MYH9--May-Hegglin Anomaly, Sebastian Syndrome, Fechtner Syndrome, and Epstein Syndrome. Inheritance is autosomal dominant. Clinical features may include high tone deafness, cataracts, leukocyte inclusions, and kidney disease leading in some cases to renal failure. Variants affecting the head domain of the Myosin-IIA protein are associated with a higher risk of nephropathy and deafness than variants affecting the tail domain (Pecci et al. 2014).

GP1BA, GP1BB, GP9--Bernard Soulier Syndrome and Platelet Type von Willebrand Disease (PT-VWD, GP1BA), and Giant Platelet Syndrome. Inheritance is autosomal recessive. Variants in GP1BA associated with PT-VWD are inherited in an autosomal dominant manner.

GATA1--X-linked thrombocytopenia. Inheritance is X-linked recessive. Clinical features include erythrocytic anemia, globin gene transcription defects, and porphyria.

Microthrombocytopenias

WAS--Wiskott-Aldrich syndrome, X-linked thrombocytopenia. Inheritance is X-linked recessive. Clinical features may include eczema, recurrent bacterial and viral infections, severe hemorrhaging, autoimmune disease such as hemolytic anemia or immune thrombocytopenic purpura, lymphomas, and X-linked Congenital Neutropenia.

Thrombocytopenias with Normal Platelet Size

CYCS--Cytochrome-C gene related thrombocytopenia. Inheritance is autosomal dominant.

ANKRD26, MASTL--Thrombocytopenia 2 (THC2) is characterized by moderately low platelet counts (family averages = 40-60/nl) (Savoia et al. 1999; Drachman et al. 2000). Platelets are of normal size. Patients often bruise easily and have moderate bleeding problems. Thrombopoietin levels are mildly elevated. Variants in ANKRD26 are associated with predisposition to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML); up to a 30 fold increase in the frequency of MDS and AML has been reported in patients with ANKRD26 gene variants (Noris et al. 2011; Noris et al. 2013; Marquez et al. 2014).

RUNX1--Familial Thrombocytopenia with Predisposition to Acute Myeloid Leukemia (AML). Inheritance is autosomal dominant. Over 40% of patients with germline variants in the RUNX1 gene develop MDS / AML at a mean age of ~ 33 years (Reviewed in Churpek et al. 2013).

MPL--Congenital Amegakaryocytic Thrombocytopenia (CAMT). Inheritance is autosomal recessive and variants in MPL have been identified in ~ 60% of patients. Clinical features include absent or reduced megakaryocytes and development of aplastic anemia and pancytopenia. AML has been reported in some patients (Ballmaier and Gerheshausen 2009).

ADAMTS13--Thrombotic Thrombocytopenic Purpura (TTP), often described as Upshaw-Schulman syndrome (USS), is a rare blood condition characterized by frequent relapses of fever, platelet thrombi in microvasculature, hemolytic anemia, consumptive thrombocytopenia, neurologic symptoms, renal disease, and possible organ failure (Levy et al. 2001). Inheritance is autosomal recessive.

See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

For this NextGen panel, the full coding regions, plus ~20 bp 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 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, undocumented and questionable variant calls are confirmed by Sanger sequencing.

Indications for Test

Patients with a family history of thrombocytopenia who may or may not have other defects including immunodeficiencies, and physical or neurological anomalies. Patients with a family history of MDS / AML. The thrombocytopenia panel may also be helpful for patients with no family history of thrombocytopenia, but who develop thrombocytopenia over time in order to help rule out a diagnosis of an inherited thrombocytopenia.

Genes

Official Gene Symbol OMIM ID
ADAMTS13 604134
ANKRD26 610855
CYCS 123970
GATA1 305371
GP1BA 606672
GP1BB 138720
GP9 173515
MASTL 608221
MPL 159530
MYH9 160775
RUNX1 151385
WAS 300392
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
MYH9-Related Disorders via the MYH9 Gene
Bernard-Soulier Syndrome via the GP1BA Gene
Bernard-Soulier Syndrome via the GP1BB Gene
Bernard-Soulier Syndrome via the GP9 Gene
Bleeding Disorders Sequencing Panel
Congenital Abnormalities of the Kidney and Urinary Tract (CAKUT) Sequencing Panel with CNV Detection
Congenital Dyserythropoietic Anemia Sequencing Panel
Nonsyndromic Hearing Loss and Deafness Sequencing Panel
Platelet Function Disorder Sequencing Panel
Severe Congenital Neutropenia Sequencing Panel
Thrombocytopenia and Predisposition to Myeloid Malignancies via the ANKRD26 Gene
Thrombocytopenia Sequencing Panel - Expanded
Thrombocytopenia via the CYCS Gene
Thrombocytopenia via the GATA1 Gene
Thrombocytopenia via the MASTL Gene
Thrombocytopenia with Predisposition to Acute Myelogenous Leukemia via the RUNX1 Gene
Thrombotic Thrombocytopenic Purpura (TTP) via the ADAMTS13 Gene
Wiskott-Aldrich Syndrome, X-linked Thrombocytopenia, and X-linked Congenital Neutropenia, via the WAS Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Balduini CL. et al. 2013. Seminars in thrombosis and hemostasis. 39: 161-71. PubMed ID: 23397552
  • Ballmaier M., Germeshausen M. 2009. British journal of haematology. 146: 3-16. PubMed ID: 19388932
  • Churpek JE. et al. 2013. Leukemia & lymphoma. 54: 28-35. PubMed ID: 22691122
  • Drachman JG. et al. 2000. Blood. 96: 118-25. PubMed ID: 10891439
  • Human Gene Mutation Database (Bio-base).
  • Levy GG. et al. 2001. Nature. 413: 488-94. PubMed ID: 11586351
  • Marquez R. et al. 2014. Leukemia & lymphoma. 55: 2945-6. PubMed ID: 24628296
  • Noris P. et al. 2011. Blood. 117: 6673-80. PubMed ID: 21467542
  • Noris P. et al. 2013. Blood. 122: 1987-9. PubMed ID: 24030261
  • Pecci A. et al. 2010. European journal of haematology. 84: 291-7. PubMed ID: 20002731
  • Pecci A. et al. 2014. Human mutation. 35: 236-47. PubMed ID: 24186861
  • Pippucci T. et al. 2011. American journal of human genetics. 88: 115-20. PubMed ID: 21211618
  • Savoia A. et al. 1999. American journal of human genetics. 65: 1401-5. PubMed ID: 10521306
Order Kits
TEST METHODS

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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