Background:
Fanconi Anemia (FA) is characterized by bone marrow failure, increased risk for cancer, and physical abnormalities. Progressive bone marrow failure is responsible for the most significant morbidity and mortality. Clinically heterogeneous, FA individuals are at increased risk for acute myelogenous leukemia, myelodysplasia, and solid tumors of the neck, head, oral cavities, and gynecological system. Congenital abnormalities are present in FA patients and include: café au lait spots or hypopigmentation; short stature; radial ray defects; eye defects such as microphthalmia; malformations of the kidney, genitalia, heart, gastrointestinal tract, ears, and feet. Currently, 15 genes corresponding to the known FA complementation groups, have been identified that, when mutated, can cause FA. Complementation testing is the current method to identify which gene is mutated in affected FA patients, followed by Sanger sequencing of the identified gene. Comprehensive next-generation sequencing techniques will eliminate the need for time intensive complementation analysis and identify the mutated FA gene with one test. Breakage analysis is still necessary to confirm the FA diagnosis.
Reasons for Referral:
- Confirmation of clinical diagnosis in patients with classical or atypical Fanconi Anemia.
- Sequence analysis of Fanconi Anemia associated genes following breakage analysis.
Methodology:
Next generation sequencing (NGS) will analyze the exons or coding regions of 15 Fanconi anemia-associated genes, as well as 2 additional genes which may be associated with the FA phenotype, using Illumina NextSeq 500 technology. Samples are prepared using hybridization probes to enrich exonic regions. This assay does not assess regions of insufficient coverage, introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and regions of high GC or polynucleotide repeats, but may contain variants that impact gene function.
Exon-level deletion/duplication analysis is performed by running the NGS data through the Genome Analysis Toolkit (GATK) Germline Copy Number Variation best practices pipeline from GATK, version 4.1.4.1. A Bayesian model was validated clinically in our lab. The model can detect copy changes at a resolution of three (3) or more probe targets (exons) for deletions and duplications in genes that do not have pseudogenes, and is not designed to detect low-level mosaicism or balanced alterations.
The 17 Fanconi anemia-associated genes are listed below:
FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP1), FANCL, FANCM, FANCM, FANCN (PALB2), FANCO (RAD51C), FANCP (SLX4), BLM, RAD51
Specimen Requirements:
- Blood: EDTA or ACD (Solution A or B):
- Adult: 5 mL
- Child: 5 mL
- Infant: 2-3 mL
- Saliva: 2 ORAgene™ Saliva Collection Kits (OGR-500) used according to manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
- Assisted Saliva: 4 ORAgene™ Assisted Saliva Collection Kits (OGR-575) used according to manufacturer instructions. Please contact KDL Client Services for an Assisted Saliva Collection Kit for patients that cannot provide a blood sample.
- Skin Fibroblast: Punch Biopsy (Cell cultures will be prepared at KDL and used for testing), or 2 T-25 confluent flasks
- DNA: 5-10µg at a minimum of 60-100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS)
- Prenatal
- Direct Amniotic Fluid (10-20mL)
- Direct CVS
- Cultured Amniocytes (2 T-25 flasks)
- Cultured CVS (2 T-25 flasks)
- Cultured Fetal Tissue: Product of Conception (2 T-25 flasks)
- Cord Blood (1-2mL)
- Notice Regarding Molecular Genetic Testing on CVS or Amniotic Fluid Specimens:
- Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood in addition to the fetal specimen. Additional charges apply for the maternal cell rule-out test.
- All genetic testing performed on Direct CVS or Amniotic Fluid specimens will be confirmed on cell cultures prepared by Knight Diagnostic Laboratories. Cell cultures will be prepared from the specimen received. Additional charges apply for confirmatory testing.
For routine testing of blood and saliva (or DNA extracted from them), KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion. For extraordinary circumstances, where testing must be performed outside of the above windows, please contact our lab. For patients with bone marrow transplants, the only acceptable specimen type is a skin biopsy (Cell cultures will be prepared at KDL before testing)
A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES. Please include detailed clinical information, including ethnicity, clinical history, and family history.
Test Performed (Days):
Weekly
Turn Around Time:
Eight Weeks
Shipment Sensitivity Requirements:
- Package and ship specimen to remain cold, but not frozen.
- Ship via overnight express, using the FedEx priority overnight label provided.
- Contact Client Services for shipping kits and instructions at (855) 535-1522.
References:
- Shimamura A, Alter B. Pathophysiology and management of inherited bone marrow failure syndromes Blood Reviews. 2010:101-122.
- Soulier J. Fanconi Anemia. American Society of Hematology Education Book. 2011(1):492-497.
Additional Info: