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Fanconi Anemia 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
1217 BRCA1 and BRCA2 81211 Add to Order
BRIP1 81479
ERCC4 81479
FANCA 81479
FANCB 81479
FANCC 81479
FANCD2 81479
FANCE 81479
FANCF 81479
FANCG 81479
FANCI 81479
FANCL 81479
FANCM 81479
PALB2 81406
RAD51 81479
RAD51C 81479
SLX4 81479
UBE2T 81479
Full Panel Price* $1990.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1217 Genes x (18) $1990.00 81211, 81406, 81479(x16) 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. If you would like to order a subset of these genes contact us to discuss pricing.

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

Over 95% of all FA patients harbor pathogenic variants in one of the 19 known FA or FA-like genes (www.fanconi.org)

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 BRCA1$690.00 81479 Add to Order
BRCA2$690.00 81479
BRIP1$690.00 81479
ERCC4$690.00 81479
FANCA$690.00 81479
FANCB$690.00 81479
FANCC$690.00 81479
FANCD2$690.00 81479
FANCE$690.00 81479
FANCF$690.00 81479
FANCG$690.00 81479
FANCI$690.00 81479
FANCL$690.00 81479
FANCM$690.00 81479
PALB2$690.00 81479
RAD51C$690.00 81479
SLX4$690.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (17) $1290.00 81479(x17) 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

Over one-third of reported pathogenic variants in the FANCA gene are large deletions or insertions; gross deletions and/or insertions have also been reported for most other FA genes, but are rare.

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Clinical Features

Fanconi Anemia (FA) is an inherited anemia associated with bone marrow failure (aplastic anemia), however the clinical features of FA can expand well beyond hematologic anomalies. FA is characterized by a range of physical abnormalities, bone marrow failure (aplastic anemia), pancytopenia, and predisposition to cancers - particularly acute myelogenous leukemia (AML), gynecologic and GI tract cancers, and cancers of the head and neck (Auerbach 2009). FA patients are up to 800 fold more susceptible to AML than the general population with a median age of onset of 13 years (Rosenberg et al. 2003). Physical abnormalities include radial ray defects (absent thumb or radius), skin pigmentation defects, short stature, microphthalmia, renal and urinary tract defects, genital defects (males in particular), gastrointestinal malformations (atresia), congenital heart disease, hearing and central nervous system defects, and general developmental delay (Tischkowitz and Hodgson 2003; Dokal 2000). About one-third of FA patients have no obvious physical abnormalities and are diagnosed only after a family member is diagnosed, or after developing hematologic anomalies such as thromobocytopenia, leukopenia, and anemia (Giampietro et al. 1997). A hallmark of FA is hypersensitivity of chromosomes to inter cross-strand linkage (ICL) agents such as diepoxybutane (DEB) or mitomycin C (MMC) (Sasaki and Tonomura 1973). Exposure of primary cell cultures from FA patients to DEB or MMC results in chromosomal aberrations (breaks, radials, rearrangements) due to damaged DNA repair mechanisms that require functional products of the Fanconi anemia genes. For example, the FANCA, -B, -C, -E, -F, -G, -L, and -M proteins are part of a nuclear core complex that regulates monoubiquitination of the FANCD2 and FANCI proteins (ID complex) during S-phase and after exposure to DNA crosslinking agents (Moldovan and D'Andrea 2009). In unaffected individuals, ubiquitination helps localize the ID complex to sites of DNA damage and facilitate repair (Grompe and van de Vrugt 2007; Smogorzewska et al. 2007), but in FA patients, this mechanism is impaired.

Genetics

FA is a genetically heterogeneous disorder. To date, 19 FA or FA-like genes have been discovered. Inheritance is primarily autosomal recessive or X-linked, however a case of heterozygous FA-like syndrome was associated with a dominant-negative variant in the RAD51 (FANCR) gene (Ameziane et al. 2015). Approximately 86% of all cases are attributed to variants in three genes: FANCA (~ 60%), FANCC (~ 16%), and FANCG (~ 10%) (Auerbach 2009). Since variants in FANCA are the most common cause of FA, it is important to note that large deletions make up over one-third of all reported pathogenic variants in FANCA. In the United States, the carrier frequency for Fanconi anemia is estimated at 1 in 181 and the incidence rate is estimated at 1 in 131,000 (http://www.fanconi.org/; Rosenberg et al. 2011). Nearly 95% of all FA cases are attributed to variants in eight genes, FANCA, -C, -G, -D1 (aka BRCA2), -D2, -E, -F, and –L that are either part of the core complex required for ID complex ubiquitination and facilitation of DNA repair or function directly in ICL recognition and repair (Grompe and van de Vrugt 2007). FA is phenotypically diverse even among related patients that harbor a common variant; null alleles however are reported to result in more severe phenotypes (Faivre et al. 2000). FA affects males and females roughly equally and affects all ethnic groups.

Testing Strategy

For this NextGen test, 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. We note that the homologous region of the FANCD2 gene is covered using Sanger sequencing and PCR primers that distinguish between the FANCD2 gene and the two FANCD2 pseudogenes. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

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

Patients with clinical features of FA, individuals with a family history of FA, and patients that develop aplastic anemia at any age, even if they present no physical abnormalities.

Genes

Official Gene Symbol OMIM ID
BRCA1 and BRCA2 600185
BRIP1 605882
ERCC4 133520
FANCA 607139
FANCB 300515
FANCC 613899
FANCD2 613984
FANCE 613976
FANCF 613897
FANCG 602956
FANCI 611360
FANCL 608111
FANCM 609644
PALB2 610355
RAD51 179617
RAD51C 602774
SLX4 613278
UBE2T 610538
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Fanconi Anemia, Complementation Group A AR 227650
Fanconi Anemia, Complementation Group B XL 300514
Fanconi Anemia, Complementation Group C AR 227645
Fanconi Anemia, Complementation Group D1 AR 605724
Fanconi Anemia, Complementation Group D2 AR 227646
Fanconi Anemia, Complementation Group E AR 600901
Fanconi Anemia, Complementation Group F AR 603467
Fanconi Anemia, Complementation Group G AR 614082
Fanconi Anemia, Complementation Group I AR 609053
Fanconi Anemia, Complementation Group J AR 609054
Fanconi Anemia, Complementation Group L AR 614083
Fanconi Anemia, Complementation Group M AR 614087
Fanconi Anemia, Complementation Group N AR 610832
Fanconi Anemia, Complementation Group O AR 613390
Fanconi Anemia, Complementation Group P AR 613951
Fanconi anemia, Complementation Group Q AR 615272
Fanconi Anemia, Complementation Group T AR 616435

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Fanconi Anemia via the FANCC Gene
Fanconi Anemia via the FANCD2 Gene
Fanconi Anemia via the FANCE Gene
Fanconi Anemia via the FANCF Gene
Fanconi Anemia via the FANCG Gene
Fanconi Anemia via the FANCI Gene
Fanconi Anemia via the FANCL Gene
Fanconi Anemia via the FANCM Gene
Fanconi Anemia via the PALB2/FANCN Gene
Fanconi Anemia via the RAD51/FANCR Gene
Fanconi Anemia via the RAD51C/FANCO Gene
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CONTACTS

Genetic Counselors
Geneticist
Citations
  • Ameziane N. et al. 2015. Nature Communications. 6: 8829. PubMed ID: 26681308
  • Auerbach A.D. 2009. Mutation Research. 668: 4-10. PubMed ID: 19622403
  • Dokal I. 2000. Bailliere's Best Practice & Research. Clinical Haematology. 13: 407-25. PubMed ID: 11030042
  • Faivre L. et al. 2000. Blood. 96: 4064–70. PubMed ID: 11110674
  • Fanconi Anemia Research Fund, Inc.
  • Giampietro P.F. et al. 1997. American Journal of Medical Genetics. 68: 58-61. PubMed ID: 8986277
  • Grompe M., van de Vrugt H. 2007. Developmental Cell. 12: 661-2. PubMed ID: 17488615
  • Moldovan G.L, D'Andrea A.D. 2009. Annual Review of Genetics. 43: 223-49. PubMed ID: 19686080
  • Rosenberg P.S. et al. 2003. Blood. 101: 822-6. PubMed ID: 12393424
  • Rosenberg P.S. et al. 2011. American Journal of Medical Genetics. Part A. 155A: 1877-83. PubMed ID: 21739583
  • Sasaki M.S., Tonomura A. 1973. Cancer Research. 33: 1829-36. PubMed ID: 4352739
  • Smogorzewska A. et al. 2007. Cell. 129: 289-301. PubMed ID: 17412408
  • Tischkowitz M.D., Hodgson S.V. 2003. Journal of Medical Genetics. 40: 1-10. PubMed ID: 12525534
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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