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Antithrombin Deficiency via the SERPINC1 Gene

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

Sequencing

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
1574 SERPINC1$780.00 81479 Add to Order
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 18 days.

Clinical Sensitivity

Clinical sensitivity should be high as two independent reports identified causative SERPINC1 mutations in 23 of 23 and 18 of 18 patients with a prior diagnosis with hereditary AD (van Boven et al. 1994; Picard et al. 2006). In a cohort of 150 patients with a prior VTE history, ~4% of individuals had mutations in the SERPINC1 gene (Fischer et al. 2013). Analytical sensitivity is ~90% as large deletions in the SERPINC1 gene have been reported in a minor subset of AD patients.

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Deletion/Duplication Testing via aCGH

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
600 SERPINC1$690.00 81479 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 Features

Antithrombin deficiency (AD) is a disorder of elevated blood clotting. An estimated 1 in 600 to 1 in 5000 individuals have AD (Harper et al. 1991; Tait et al. 1994). While many patients are asymptomatic until later in life, individuals with AD are at heightened risks for venous thromboembolism (VTE). VTE is a major cause of morbidity and death in hospitalized patients (Heit et al. 2001) and maternal death in pregnancy (James et al. 2013). About half of people with AD will develop at least one abnormal blood clot during their lifetime, often after adolescence. In severe cases clotting may occur during childhood. Environmental factors including surgery, increased age, and immobility heighten risk for VTE.

Antithrombin protein regulates blood clotting by inactivating factors that facilitate clotting such as thrombin. There are two types of AD. Type I AD deficiency is characterized by loss of both AD protein activity and levels within the blood. Type II, the more common form, presents with normal protein levels, but impaired activity. AD concentrates, low molecular weight heparin, and other anticoagulant therapies have been used to treat disease. Diagnosis can be difficult as commercial kits assaying antithrombin activity vary widely (Javela et al. 2013), and secondary pathologies such as sepsis result in acquired AD (White and Perry 2001).

Genetics

Antithrombin deficiency is inherited in an autosomal dominant manner with variable penetrance cause by mutations in the SERPINC1 gene. Risk of VTE development is highly correlated to degree of antithrombin loss of function. Type I deficiency is primarily caused through nonsense or small insertion/deletion mutations resulting in frameshift and premature protein termination (Cooper et al. 2011). Missense mutations altering glycosylation and leading to intracellular retention of antithrombin have also been described in individuals with type I deficiency (Fitches et al. 2001; Beauchamp et al. 1998). Type II deficiency is primarily caused by missense mutations disrupting enzyme-inhibitor and heparin binding sites, although mutations are found throughout the coding region. Large deletions are found in <10% of individuals with type I deficiency. Patients homozygous and compound heterozygous for AD defects have been reported in a small number of cases. Such mutations are associated with severe thromboembolic disease (Kuhle et al. 2001; Picard et al. 2010). Complete deficiency in antithrombin is incompatible with life (Ishiguro et al. 2000). Antithrombin is a serine protease inhibitor found freely in blood plasma. It inactivates pro-coagulation proteins, primarily thrombin and factor X, to prevent excessive clot formation and is a primary factor for dissolution of clots after vessel repair.

Testing Strategy

Our DNA sequencing test involves bidirectional Sanger sequencing of the entire SERPINC1 gene plus ~20 bp of flanking non-coding DNA on either side of each exon. 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

Patients with family history of VTE and pregnancy loss are candidates for testing. Ideal candidates have diagnostic data indicating impaired antithrombin function or levels. Genetic testing is a strong tool to distinguish between acquired and inherited forms of AD (Cooper et al. 2011; Patnaik and Moll 2008).

Gene

Official Gene Symbol OMIM ID
SERPINC1 107300
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Antithrombin III Deficiency 613118

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Beauchamp NJ, Pike RN, Daly M, Butler L, Makris M, Dafforn TR, Zhou A, Fitton HL, Preston FE, Peake IR, Carrell RW. 1998. Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation. Blood 92: 2696–2706. PubMed ID: 9763552
  • Cooper PC, Coath F, Daly ME, Makris M. 2011. The phenotypic and genetic assessment of antithrombin deficiency. Int J Lab Hematol 33: 227–237. PubMed ID: 21401902
  • Fischer R, Sachs UJ, Heidinger KS, Eisenburger D, Kemkes-Matthes B. 2013. Prevalence of hereditary antithrombin mutations is higher than estimated in patients with thrombotic events. Blood Coagul. Fibrinolysis 24: 444–448. PubMed ID: 23429250
  • Fitches AC, Lewandowski K, Olds RJ. 2001. Creation of an additional glycosylation site as a mechanism for type I antithrombin deficiency. Thromb. Haemost. 86: 1023–1027. PubMed ID: 11686319
  • Harper PL, Luddington RJ, Daly M, Bruce D, Williamson D, Edgar PF, Perry DJ, Carrell RW. 1991. The incidence of dysfunctional antithrombin variants: four cases in 210 patients with thromboembolic disease. Br. J. Haematol. 77: 360–364. PubMed ID: 2012760
  • Heit JA, Melton LJ 3rd, Lohse CM, Petterson TM, Silverstein MD, Mohr DN, O’Fallon WM. 2001. Incidence of venous thromboembolism in hospitalized patients vs community residents. Mayo Clin. Proc. 76: 1102–1110. PubMed ID: 11702898
  • Ishiguro K, Kojima T, Kadomatsu K, Nakayama Y, Takagi A, Suzuki M, Takeda N, Ito M, Yamamoto K, Matsushita T, Kusugami K, Muramatsu T, et al. 2000. Complete antithrombin deficiency in mice results in embryonic lethality. J. Clin. Invest. 106: 873–878. PubMed ID: 11018075
  • James AH, Konkle BA, Bauer KA. 2013. Prevention and treatment of venous thromboembolism in pregnancy in patients with hereditary antithrombin deficiency. Int J Womens Health 5: 233–241. PubMed ID: 23662090
  • Javela K, Engelbarth S, Hiltunen L, Mustonen P, Puurunen M. 2013. Great discrepancy in antithrombin activity measured using five commercially available functional assays. Thromb. Res. 132: 132–137. PubMed ID: 23768451
  • Kuhle S, Lane DA, Jochmanns K, Male C, Quehenberger P, Lechner K, Pabinger I. 2001. Homozygous antithrombin deficiency type II (99 Leu to Phe mutation) and childhood thromboembolism. Thromb. Haemost. 86: 1007–1011. PubMed ID: 11686316
  • Patnaik MM, Moll S. 2008. Inherited antithrombin deficiency: a review. Haemophilia 14: 1229–1239. PubMed ID: 19141163
  • Picard V, Chen J-M, Tardy B, Aillaud M-F, Boiteux-Vergnes C, Dreyfus M, Emmerich J, Lavenu-Bombled C, Nowak-Göttl U, Trillot N, Aiach M, Alhenc-Gelas M. 2010. Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency. Hum. Genet. 127: 45–53. PubMed ID: 19760264
  • Picard V, Nowak-Göttl U, Biron-Andreani C, Fouassier M, Frere C, Goualt-Heilman M, Maistre E de, Regina S, Rugeri L, Ternisien C, Trichet C, Vergnes C, et al. 2006. Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene. Hum. Mutat. 27: 600. PubMed ID: 16705712
  • Tait RC, Walker ID, Perry DJ, Islam SI, Daly ME, McCall F, Conkie JA, Carrell RW. 1994. Prevalence of antithrombin deficiency in the healthy population. Br. J. Haematol. 87: 106–112. PubMed ID: 7947234
  • van Boven HH, Olds RJ, Thein SL, Reitsma PH, Lane DA, Briët E, Vandenbroucke JP, Rosendaal FR. 1994. Hereditary antithrombin deficiency: heterogeneity of the molecular basis and mortality in Dutch families. Blood 84: 4209–4213. PubMed ID: 7994035
  • White B, Perry D. 2001. Acquired antithrombin deficiency in sepsis. Br. J. Haematol. 112: 26–31. PubMed ID: 11167778
Order Kits
TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

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 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 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 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

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).

Analytical Limitations

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 20 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.

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
  • The first four pages of the requisition form must accompany all specimens.
  • Billing information is on the third and fourth pages.
  • Specimen and shipping instructions are listed on the fifth and sixth pages.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
  • If refrigerated, blood specimen is good for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • NextGen Sequencing Tests: Send in screw cap tube at least 10 µg of purified DNA at a concentration of at least 50 µg/ml
  • Sanger Sequencing Tests: Send in a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml. For tests involving the sequencing of more than three genes, send an additional 5 µg DNA per gene. DNA may be shipped at room temperature.
  • Deletion/Duplication via aCGH: Send in screw cap tube at least 1 µg of purified DNA at a concentration of at least 100 µg/ml.
  • Whole-Genome Chromosomal Microarray: Collect at least 5 µg of DNA in TE (10 mM Tris-cl pH 8.0, 1mM EDTA), dissolved in 200 µl at a concentration of at least 100 ng/ul (indicate concentration on tube label). DNA extracted using a column-based method (Qiagen) or bead-based technology is preferred.

CELL CULTURE

(Delivery accepted Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Ship at least two T25 flasks of confluent cells.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We do not culture cells.