Background:
Inherited bone marrow failure syndromes are a group of disorders characterized by the inability to sufficiently produce red blood cells, white blood cells or platelets. These conditions are caused by mutations in genes involved in ribosome assembly, DNA repair, and telomere maintenance and are associated with Fanconi anemias, Diamond Blackfan anemia, Shwachman Diamond syndrome, dyskeratosis congentia, aplastic anemias, and myelodysplastic anemia syndromes. This next-generation sequencing tests the coding regions for117 genes that are associated with these conditions. These may be inherited as autosomal recessive, dominant or X-linked pattern.
Reasons for Referral:
- Genetic diagnosis for patients with bone marrow failure
- Carrier testing
Methodology:
Genomic DNA is analyzed using next-generation sequencing (NGS) on the Illumina NextSeq 2000 platform, with target enrichment performed using hybridization-based probes to capture exonic (coding) regions of the gene(s). Single nucleotide variants (SNVs) and small insertions or deletions (INDELs) are identified using the Illumina DRAGEN Enrichment Workflow, executed onboard the NextSeq2000. This pipeline combines software and hardware acceleration to generate high-confidence germline haplotype calls. Clinical and analytical validation of DRAGEN was performed in our laboratory. Based on validation study results, for SNVs, this assay achieves >96% analytical sensitivity and >99% positive predictive value (PPV). For INDELs <50 bp, the analytical sensitivity is >87% and the PPV is >97%. INDELs >50 bp may be detected but the sensitivity for these is reduced.
Exon-level copy number variants (CNVs) are detected using the Germline Copy Number Variation Best Practices pipeline from GATK. A Bayesian model, clinically validated in our laboratory, enables detection of deletions and duplications involving three or more contiguous exons in genes with adequate probe coverage and without complicating factors (e.g. pseudogene homology, short tandem repeats, segmenral duplications). Please note that exon-centric microarray remains the gold standard for exonic copy number variant calling. If exon-centric microarray is of interest, please contact the laboratory for additional information.
This test is not designed to detect polynucleotide repeats, low-level mosaicism, structural rearrangements or balanced alterations (e.g. inversions, gene conversion events, translocations, etc.) or variants in difficult regions. Additionally, variants located in regions of insufficient coverage, including introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and of high GC content may not be detected. This test does not provide complete coverage of all exons and noncoding regions may have limited information and ability to interpret. Variants in introns that are greater than 10 bp from the intron-exon junction may be analyzed. Please contact the laboratory if interrogation of intronic sequence greater than 10 bp from the intron-exon boundary is desired.
The 117 Bone Marrow Failure-associated genes are listed below:
ABCB7, ACD, AK2, ALAS2, ANKRD26, ATM, ATR, BLM, BRCA1, BRCA2, BRIP1, c6orf25 (MPIG6B), CBL, CDAN1, CECR1 (ADA2), CLPB, CSF3R, CTC1, CXCR4, DDX41, DKC1, DNAJC21, EFTUD1 (EFL1), ELANE, EPO, ERCC4, ERCC6L2, ETV6, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, G6PC3, GATA1, GATA2, GFI1, HAX1, HOXA11, ITK, JAGN1, KLF1, KRAS, LIG4, LYST, MAD2L2, MECOM, MPL, MYSM1, NBN, NHP2, NOP10, NRAS, PALB2, PARN, PRF1, PTPN11, PUS1, RAB27A, RAC2, RAD51, RAD51C, RBM8A, RFWD3, RPL11, RPL15, RPL18, RPL26, RPL27, RPL31, RPL35, RPL35A, RPL5, RPL9, RPS10, RPS15, RPS15A, RPS19, RPS24, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS7, RTEL1, RUNX1, SAMD9, SAMD9L, SBDS, SLC37A4, SLX4, SMARCD2, SRP54, SRP72, TBXAS1, TCN2, TERC, TERT, THPO, TINF2, TP53, TSR2, UBE2T, USB1, VPS45, WAS, WDR1, WIPF1, WRAP53, XRCC2, ZCCHC8
For a comprehensive list of regions with limited coverage on this panel, please refer to the following table.
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 Kit(s) (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 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.
Buccal Cells: 4 CytoSoft™ Cytology Brush (Medical Packaging CYB-1) used according to manufacturer instructions. Please contact KDL Client Services for a Buccal 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: 7-10 µg 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).
Important note on specimen type: This test is designed for detecting germline variants. As such, results may be compromised for blood, bone marrow, or saliva from patients with certain hematological conditions that are in an active disease state. In such cases, skin fibroblasts are highly recommended.
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 within 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:
3-4 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:
- Haematologica. 2010 Aug; 95(8): 1236–1240
Additional Info: