Diagnosis of Fanconi Anemia by Diepoxybutane Analysis

Arleen D. Auerbach1

1 Program in Human Genetics and Hematology, The Rockefeller University, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.7
DOI:  10.1002/0471142905.hg0807s85
Online Posting Date:  April, 2015
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Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck. The main role of FA proteins is in the repair of DNA interstrand crosslinks (ICLs). FA results from pathogenic variants in at least sixteen distinct genes, causing genomic instability. Although the highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, diagnosis based on a profound sensitivity to DNA‐crosslinking agents can be used to identify the pre‐anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false‐positive and false‐negative results. © 2015 by John Wiley & Sons, Inc.

Keywords: Fanconi anemia; DEB test; DNA interstrand crosslink repair; genomic instability

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Table of Contents

  • Introduction
  • Basic Protocol 1: Diepoxybutane Test for Postnatal Diagnosis of Fanconi Anemia
  • Support Protocol 1: Working with and Disposing of Diepoxybutane
  • Support Protocol 2: Giemsa Staining for Chromosome‐Breakage Analysis
  • Alternate Protocol 1: Diepoxybutane Test Using Fibroblast Cultures
  • Alternate Protocol 2: Diepoxybutane Test for Prenatal Diagnosis of Fanconi Anemia
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Diepoxybutane Test for Postnatal Diagnosis of Fanconi Anemia

  • Peripheral blood: collect in a preservative‐free sodium heparin Vacutainer tube (e.g., Fisher Scientific)
  • Complete RPMI/15% FBS (see appendix 3G)/1% (w/v) phytohemagglutinin (PHA; Sigma‐Aldrich or Thermo Scientific)
  • PBS ( appendix 2D)
  • Diepoxybutane (1,3‐butadiene diepoxide, DEB; store at 4°C; Sigma‐Aldrich)
  • 1 μg/ml colcemid solution in PBS (store at 4°C; e.g., Life Technologies)
  • 0.075 M potassium chloride (KCl), 37°C
  • Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)
  • 10‐ml syringe equipped with 18.5‐G needle
  • 25‐cm2 tissue culture flasks
  • 15‐ml sterile conical‐bottom centrifuge tubes with caps
  • IEC clinical centrifuge or equivalent
  • Microscope slides (store in 70% ethanol)
  • Inverted microscope
  • Additional reagents and equipment for collection of peripheral blood lymphocytes (unit 4.1, Bangs and Donlon, ), tissue culture ( appendix 3G), preparing metaphase chromosome slides (unit 4.1, Bangs and Donlon, ), and karyotyping ( appendix 4A)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Working with and Disposing of Diepoxybutane

  • Gurrs buffer tablets, pH 6.8 (Bio/medical Specialties)
  • Giemsa stain (e.g., Life Technologies)
  • Metaphase chromosome slide (see protocol 1Basic Protocol)
  • Permount histological mounting medium
  • Coplin jars

Support Protocol 2: Giemsa Staining for Chromosome‐Breakage Analysis

  Additional Materials (also see protocol 1Basic Protocol)
  • Tissue biopsies (e.g., full‐thickness skin sample taken using a 3‐mm punch)
  • Complete DMEM/20% (v/v) FBS ( appendix 3G)
  • 0.051 M (0.38% w/v) potassium chloride (KCl)
  • 1× trypsin/EDTA solution ( appendix 3G)
  • 60‐mm tissue culture plates
  • Disposable scalpels
  • Additional reagents and equipment for passaging cells by trypsinization and counting cells ( appendix 3G)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Diepoxybutane Test Using Fibroblast Cultures

  Additional Materials (also see protocol 1Basic Protocol)
  • Fetal cells, enough to establish cultures in two 25‐cm2 flasks
  • Growth medium: 1:1 mixture of Chang medium (unit 8.4, Minehart Miron, ) and complete RPMI/15% FBS ( appendix 3G)
  • Additional materials and equipment for culture of chorionic villi (unit 8.3, Breman and Patel, ) and amniotic fluid (unit 8.4, Minehart Miron, )
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Key References
  Auerbach, A.D., Rogatko, A., and Schroeder‐Kurth, T.M. 1989. See above.
  Presents evidence from the International Fanconi Anemia Registry showing that the DEB test is a useful discriminator for FA.
  Giampietro, P.F., Davis, J.G., Adler‐Brecher, B., Verlander, P.C., Auerbach, A.D., and Pavlakis, S.G. 1993. The need for more accurate and timely diagnosis in Fanconi anemia: A report from the International Fanconi Anemia Registry. Pediatrics 91:1116‐1120.
  Discusses the need for clinicians to have increased awareness of the great variability in the FA phenotype and the need for ordering DEB testing to rule out a diagnosis of FA in pre‐anemic patients and patients without typical malformations.
  Auerbach, A.D. 2009. Fanconi anemia and its diagnosis. Mutat. Res. 668:4‐10.
  Presents an overview of current knowledge regarding the varied phenotypic manifestations of FA and procedures for diagnosis based upon abnormal DNA damage responses.
Internet Resources
  Fanconi Anemia Mutation Database.
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