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Congenital Fibrinogen Deficiency via the FGB Gene

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

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
6985 FGB$690.00 81479 Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

For Sanger Sequencing click here.
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

Rare bleeding disorders (RBD) are comprised of inherited deficiencies of coagulation factors fibrinogen, FII, FV, FV + FVIII, FVII, FX, FXI, and FXIII. CFDs are found ~8% of all RBD cases (Peyvandi et al. 2013). In patients with CFD, causative FGB mutations are found in ~15% of cases (Hanss and Biot 2001). Analytical sensitivity is >95% as the majority of causative variants are detectable by sequencing. Large deletions in the FGB gene have been reported in afibrinogenemia (Liu et al. 1985).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FGB$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

Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder, affecting about 1 in a million people, with wide variability in clinical presentation from asymptomatic to life-threatening bleeds. CFDs can be subdivided into type I (afibrinogenemia and hypofibrinogenemia) and type II deficiencies (dysfibrinogenemia and hypo-dysfibrinogenemia). Type I deficiencies are defined by individuals having reduced activity and levels of fibrinogen whereas type II individuals have normal fibrinogen levels but impaired function (Acharya and Dimichele 2008). Afibrinogenemia, the most severe form of CFD, typically presents in the neonatal period with umbilical cord bleeding being the most characteristic of disease. Bleeding tendencies are variable but include life-threatening spontaneous and trauma related bleeds. Patients with hypofibrinogenemia have a milder disease course, as loss of fibrinogen protein is less severe than individuals with afibrinogenemia. Bleeding episodes in these individuals occur later in life often after trauma or surgery. Patients with dysfibrinogenemia are primarily asymptomatic but may experience bleeding after trauma or child birth (de Moerloose et al. 2013). Unlike type I deficiencies, individuals with type II deficiencies have been reported to be at increased risk of thrombosis (Morris et al. 2009). Acquired fibrinogen deficiencies have been found in individuals with liver disease and autoantibodies (Kujovich 2005 ; Dear et al. 2007). Genetic testing is helpful in differential diagnosis of other rare bleeding disorders, distinguishing inherited and acquired forms, and for diagnosis of asymptomatic hypofibrinogenemia and dysfibrinogenemia patients prior to surgery. Treatment options include fibrinogen concentrates, cryoprecipitate, and fresh frozen plasma (Acharya and Dimichele 2008).

Genetics

CFD is caused through mutations in the FGA, FGB, or FGG genes. Together these genes encode the hexameric glycoprotein fibrinogen. Onset of afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia can occur through mutations in any of the three fibrinogen genes. Afibrinogenemia is inherited in an autosomal recessive manner with null mutations accounting for the majority of causative variants. Hypofibrinogenemia and dysfibrinogenemia are inherited in autosomal dominant manner with reduced disease penetrance predominantly due to missense mutations (de Moerloose et al. 2013). CFD severity is directly correlative to degree of impaired fibrinogen level and function. Causative mutations for afibrinogenemia and hypofibrinogenemia can overlap with patients homozygous for the mutation presenting with afibrinogenemia. Thus, asymptomatic individuals with hypofibrinogenemia often are carriers for afibrinogenemia (Acharya and Dimichele 2008). Mutations in the FGB gene account for ~15% of CFD cases with missense mutations being most prevalent and affecting assembly, secretion, and/or stability of the hexameric fibrinogen (Matsuda and Sugo 2002; Hanss and Biot 2001; Vu and Neerman-Arbez 2007). Large deletions in the FGB gene have only been reported once (Liu et al. 1985). Fibrinogen is synthesized in the liver as a disulphide linked hexamer comprised of two heterotrimers consisting of one alpha, beta, and gamma chain. Fibrinogen is catalyzed into fibrin by thrombin to promote blood clot formation through platelet bridging (Acharya and Dimichele 2008).

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. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and 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.

This test provides full coverage of all coding exons of the FGB gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

Candidates have decreased levels of fibrinogen antigen and activity (less than 0.5 g L-1) for type I CFD. Type II individuals present with discrepancies between antigen and activity measurements. All coagulation tests that depend on fibrin as an end point, PT, PPT, TT, and reptilase times are typically prolonged. Patients with a family history of hypofibrinogenemia and dysfibrinogenemia are ideal candidates for testing (Acharya and Dimichele 2008).

Gene

Official Gene Symbol OMIM ID
FGB 134830
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Afibrinogenemia, congenital 202400

Related Tests

Name
Bleeding Disorders Sequencing Panel
Coagulation Factor Deficiency Sequencing Panel
Congenital Fibrinogen Deficiency Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Acharya SS, Dimichele DM. 2008. Rare inherited disorders of fibrinogen. Haemophilia 14: 1151–1158. PubMed ID: 19141154
  • de Moerloose P, Casini A, Neerman-Arbez M. 2013. Congenital fibrinogen disorders: an update. Semin. Thromb. Hemost. 39: 585–595. PubMed ID: 23852822
  • Dear A, Brennan SO, Sheat MJ, Faed JM, George PM. 2007. Acquired dysfibrinogenemia caused by monoclonal production of immunoglobulin lambda light chain. Haematologica 92: e111–117. PubMed ID: 18024387
  • Hanss M, Biot F. 2001. A database for human fibrinogen variants. Ann. N. Y. Acad. Sci. 936: 89–90. PubMed ID: 11460527
  • Kujovich JL. 2005. Hemostatic defects in end stage liver disease. Crit Care Clin 21: 563–587. PubMed ID: 15992673
  • Liu CY, Koehn JA, Morgan FJ. 1985. Characterization of fibrinogen New York 1. A dysfunctional fibrinogen with a deletion of B beta(9-72) corresponding exactly to exon 2 of the gene. J. Biol. Chem. 260: 4390–4396. PubMed ID: 3156856
  • Matsuda M, Sugo T. 2002. Structure and function of human fibrinogen inferred from dysfibrinogens. Int. J. Hematol. 76 Suppl 1: 352–360. PubMed ID: 12430881
  • Morris TA, Marsh JJ, Chiles PG, Magaña MM, Liang N-C, Soler X, Desantis DJ, Ngo D, Woods VL Jr. 2009. High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension. Blood 114: 1929–1936. PubMed ID: 19420351
  • Peyvandi F. et al. 2013. Blood. 122: 3423-31. PubMed ID: 24124085
  • Vu D, Neerman-Arbez M. 2007. Molecular mechanisms accounting for fibrinogen deficiency: from large deletions to intracellular retention of misfolded proteins. J. Thromb. Haemost. 5 Suppl 1: 125–131. PubMed ID: 17635718
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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