(performed only on a research basis)

Instructions for Collection and Shipment of Blood Samples for Diepoxybutane (DEB) Test to Rule Out Fanconi Anemia.

DEB requisition form for peripheral blood testing

Specimen Requirements of Fibroblasts for DEB Test to Rule out Fanconi Anemia

Shipping Instructions of Fibroblasts for DEB Test to rule out Fanconi Anemia

THE DIEPOXYBUTANE (DEB) TEST TO RULE OUT FANCONI ANEMIA (FA)

Fanconi anemia (FA) is a recessively inherited syndrome characterized by progressive pancytopenia and a variety of birth defects in some patients. Although thirteen FA genes have been isolated, the molecular pathophysiology of FA remains poorly understood. Hypersensitivity to the clastogenic effect of DNA crosslinking agents provides a unique marker for the diagnosis of FA. This cellular characteristic can be used to identify the pre-anemic phase patient as well as the patient with aplastic anemia and/or leukemia who may or may not have the classic physical stigmata associated with FA. A variety of methods are available for testing crosslink sensitivity including diepoxybutane (DEB) and mitomycin C (MMC). Our experience has shown that for FA diagnosis, DEB is more reliable then MMC, which may have a higher rate of false positives and false negatives.

The clinical phenotype in FA is extremely variable making diagnosis on a clinical basis alone difficult and unreliable. It is extremely important to ascertain whether VATER or VACTERL patients have FA, so that the family can receive accurate and timely genetic counseling regarding reproductive options. It is also essential that aplastic anemia patients be tested to rule out FA, as optimal treatment regimens are tailored to diagnosis.

METHODS

Cell Culture

Peripheral blood is stimulated with PHA and cultured for 72-96 hours. Cultures are set up in duplicate for DEB studies and a replicate set of cultures is established to serve as untreated controls. DEB is added to the cultures 24 hours after their initiation, at a final concentration in the medium of 0.1 ug/ml, thus exposing the cells to the chemical for 48-72 hours. At this concentration of DEB, FA cells exhibit multiple chromatid breaks and exchanges while there is little clastogenic effect on normal cells. The induction of this type of chromosome breakage by DEB thus appears to be a unique marker for FA and is used to distinguish the FA patient from other patients.

Chromosome Breakage Analysis

Metaphase spreads are prepared by standard cytogenetic methods. Analysis is performed on 50-100 Giemsa stained metaphases from each DEB-treated preparation (20-25 metaphases in the case of very high breakage). If chromosomal breakage is increased above the normal range an untreated preparation is analyzed. Each cell is scored for chromosome number and for the numbers and types of structural abnormalities. The mean baseline chromosomal breakage frequency in peripheral blood lymphocytes ranges from 0.00-0.05 breaks per cell in normal control individuals. The range is from 0.02-0.85 per cell in FA patients. Note that the baseline breakage in some FA patients does not differ from normal controls and some FA patients would be misdiagnosed if only baseline breakage studies were considered. The mean DEB-induced chromosomal breakage frequency (at 0.1mg/ml DEB) for normal control individuals ranges from 0.00-0.10 breaks per cell, for the FA patients from 1.06-23.9 breaks per cell. There is thus no overlap in the range for the FA group compared with control groups. It is important to note that in DEB-cultures from FA affected individuals we have found that the percent of cells exhibiting chromosomal breakage can vary from as low as 10% to as high as 100%. The finding of a consistent population of DEB-resistant cells is a result of genetic reversion; these patients are said to exhibit somatic mosaicism in their lymphocytes.

DEB TEST REFERENCES

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Auerbach AD, Warburton D, Bloom AD, Chaganti RSK: Prenatal detection of the Fanconi anemia gene by cytogenetic methods. Am J Hum Genet 31:77-81, 1979.

Auerbach AD, Adler B, Chaganti RSK: Prenatal and postnatal diagnosis and carrier detection of Fanconi anemia by a cytogenetic method. Pediatr 67:128-135, 1981.

Auerbach AD, Sagi M, Adler B: Fanconi anemia: Prenatal diagnosis in thirty fetuses at risk. Pediatr 76:794-800, 1985.

Auerbach AD, Zhang M, Ghosh R, Pergament E, Verlinsky Y, Nicolas H, Boue J: Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia. Human Genet 73:86-88, 1986.

Auerbach AD, Rogatko A, and T Schroeder-Kurth, International Fanconi Anemia Registry: Relation of Clinical Symptoms to Diepoxybutane Sensitivity. Blood 73: 391-396, 1989.

Auerbach AD, Ghosh R, Pollio PC, Zhang M: Diepoxybutane (DEB) test for prenatal and postnatal diagnosis of Fanconi anemia. In: Fanconi anemia, Clinical, Cytogenetic and Experimental Aspects, Schroeder TM, Auerbach AD, Obe G (eds), Heidelberg, Springer-Verlag, pp.71-82, 1989.

Auerbach AD, Prenatal and Postnatal Diagnosis in Aplastic Anemia. in: Methods in Hematology, Vol. 21 Perinatal Hematology ed. Alter BP, Churchill Livingstone, pp.236-240, 1989.

Auerbach AD: Cytogenetics in constitutional aplastic anemia. In: Aplastic Anemia and Other Bone Marrow Failure Syndromes, NT Shahidi, (ed). Springer-Verlag, New York pp.51-62, 1990.

Auerbach AD, Liu Q, Ghosh R, Pollack MS, Douglas GW, Broxmeyer HE: Prenatal identification of potential donors for umbilical cord blood transplantation for Fanconi anemia. Transfusion 30:682-687, 1990.

Giampietro PF, Adler-Brecher B, Verlander PC, Pavlakis SG, Davis JG, Auerbach AD: The need for more accurate and timely diagnosis in Fanconi anemia. A report from the International Fanconi Anemia Registry. Pediatrics: 91:1116-1120, 1993.

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Giampietro PF, Verlander PC, Davis JG, Auerbach AD. Diagnosis of Fanconi anemia in patients without congenital malformations: An International Fanconi Anemia Registry Study. Am J Med Genet 68:58-61, 1997.

Gregory JJ, Wagner JE, Verlander PC, Levran O, Batish SD, Eide C, Steffenhagen A, Hirsch B, Auerbach AD. Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lympho-hematopoietic stem cells. Proc Natl Acad Sci (USA) 98:2532-2537, 2001.

Auerbach AD: Diagnosis of Fanconi anemia by Diepoxybutane Analysis. In: Current Protocols in Human Genetics, Dracopoli NC, Haines JL, Korf BR, Moir DT, Morton CC, Seidman CE, Seidman JG, Smith DR (eds), Hoboken, NJ, John Wiley & Sons, Inc., Supplement 37, pp 8.7.1-8.7.15, 2003.