Letter to the Editor

Confounding Factors in the Diagnosis ofFanconi Anaemia

To the Editor:

The letter by O.W.J. Quarrell et al. [1998] on ‘‘BallerGerold Syndrome and Fanconi Anaemia’’ is concernedwith false-negative results from chromosomal break-age studies that should confirm or exclude the diagno-sis of Fanconi anaemia (FA). They present a case testedfor chromosomal breakage at 3 and 16 days and 16months after birth, with several protocols being usedand only the last test (with mitomycin C as a clastogen)being conclusively positive. The authors cite a true FAcase, i.e., with pathogenic mutations in the FAC gene,who nevertheless scored negatively in the chromosom-al breakage test [Dokal et al., 1996] and infer thatnegative cytogenetic test results may not always beinformative. This is a discomforting conclusion, sinceFA is clinically variable and many patients suspectedof having FA do score negatively in the test. Do wereally need to question all these negative test results?

Based on many years of experience with diagnosisand research on FA we think that the test is essentiallyreliable, but there are indeed some caveats.

It is generally accepted that true FA patients arecharacterized by a striking constitutional hypersensi-tivity to cross-linking agents [Sasaki and Tonomura,1973; Auerbach 1990; Auerbach et al., 1998, and refer-ences therein; Giampietro et al., 1993], but the meth-odology used to demonstrate this cellular phenotypevaries among laboratories. Diepoxybutane and mito-mycin C are most commonly used as the clastogen, ni-trogen mustard, cisplatinum, and trenimon being lesscommon. Although exposure conditions may vary con-siderably, chromosomal breakage is most commonlyused as an endpoint, while flow cytometric assessmentof G2 phase arrest is also used [Seyschab et al., 1995].Because FA is rare (in the order of five per millionbirths) many cytogenetic laboratories may not encoun-ter an FA patient for many years. Thus, positive con-trols and other laboratory routines may be inadequate.This situation has made FA diagnosis prone to errors,discrepancies, and inconsistencies. Concentration of

FA diagnostics in fewer specialized centers would prob-ably improve this situation.

A second caveat is reverse mosaicism, which is notinfrequent in FA [Kwee et al., 1983; Auerbach, 1990; LoTen Foe et al., 1997]. Mosaic FA patients apparentlyhave two populations of lymphocytes, one being hyper-sensitive to the clastogen whereas the other is normal.Depending on the specific protocol used, patients witha high proportion of reverted cells may be falsely diag-nosed as negatives. Studying skin biopsies has helpedto settle the diagnosis in such cases, since fibroblastsare more likely to retain their clastogen hypersensitiv-ity [Arwert and Kwee, 1989]. Phenotypic reversion tonormal has been correlated with intragenic homolo-gous recombination in compound heterozygous pa-tients giving rise to the segregation of a wildtype alleleat the disease locus [Lo Ten Foe et al., 1997]. The re-sulting cell is thus essentially cured from the diseasetrait. Progeny from a reverted stem cell is expected tohave a proliferative advantage over affected cells andthus may gradually expand and take over hematopoi-esis. We have observed full-fledged FA patients (i.e.,with no apparent sign of mosaicism) changing into ap-parent false-negatives within a period of several years[Lo Ten Foe et al., 1997; H. Hoehn, unpublished obser-vations], presumably as a result of such progressivechanges in the hematopoietic progenitor or stem cellpopulation. Without the initial positive result such pa-tients would be incorrectly diagnosed as ‘‘non-FA’’when tested on blood only; however, skin fibroblastswould still allow us to demonstrate their constitutionalhypersensitivity. Thus, the occurrence of somatic mo-saicism seriously complicates the correct diagnosis ofblood-test–negative patients and calls for supplemen-tary fibroblast studies.

In summary, we think that in spite of some well-defined complications associated with the chromosom-al breakage test, the proposal of Dr. Quarrell et al. thata diagnosis based on clinical symptomatology shouldsometimes overrule negative cytogenetic test resultsshould not be implemented. In cases where FA orBaller Gerold syndrome are strongly suspected, rigor-ous testing (including the use of skin fibroblasts) isrecommended. The diagnosis of Baller Gerold syn-drome should only be accepted when fibroblasts arenegative for FA.

*Correspondence to: Hans Joenje, Department of Human Ge-netics, Medical Centre Free University, Van der Boechorststraat7, NL-1081 BT Amsterdam, The Netherlands.

Received 10 March 1998; Accepted 11 March 1998

American Journal of Medical Genetics 79:403–404 (1998)

© 1998 Wiley-Liss, Inc.

REFERENCESArwert F, Kwee ML (1989): Chromosomal breakage in response to cross-

linking agents in the diagnosis of Fanconi anaemia. In Schroeder-Kurth TM, Auerbach AD, Obe G (eds): ‘‘Fanconi Anaemia, Clinical,Cytogenetic and Experimental Aspects.’’ Berlin: Springer Verlag, pp83–92.

Auerbach AD (1990): Cytogenetics in constitutional aplastic anemia: InShahidi NT (ed): ‘‘Aplastic Anemia and Other Bone Marrow FailureSyndromes.’’ New York: Springer-Verlag, pp 51–62.

Auerbach AD, Buchwald M, Joenje H (1998): Fanconi anemia. In Vogel-stein B, Kinzler WK (eds): ‘‘The Genetic Basis of Human Cancer.’’ NewYork: McGraw–Hill, pp 317–332.

Dokal I, Chase A, Morgan NV, Coulthard S, Hall G, Mathew CG, RobertsI (1996): Positive diepoxybutane test in only one of two brothers foundto be compound heterozygotes for Fanconi’s anaemia complementationgroup C mutations. Br J Haematol 93:813–816.

Giampietro PF, Adler-Brecher B, Verlander PC, Pavlakis SG, Davis JG,Auerbach AD (1993): The need for more accurate and timely diagnosisin Fanconi anemia—A report from the International Fanconi AnemiaRegistry. Pediatrics 91:1116–1120.

Kwee ML, Poll EHA, Van de Kamp JJP, De Koning H, Eriksson AW, JoenjeH (1983): Unusual response to bifunctional alkylating agents in a caseof Fanconi anaemia. Hum Genet 64:384–387.

Lo Ten Foe JR, Kwee ML, Rooimans MA, Oostra AB, Veerman AJP, PauliRM, Shahidi NT, Dokal I, Roberts I, Altay C, Gluckman E, Gibson RA,Mathew C, Arwert F, Joenje H (1997): Somatic mosaicism in Fanconianemia: Molecular basis and clinical significance. Eur J Hum Genet5:137–148.

Quarrell OWJ, Maltby EL, Harrison CJ (1998): Baller Gerold syndromeand Fanconi anemia. Am J Med Genet 75:228–229.

Sasaki MS, Tonomura A (1973): A high susceptibility of Fanconi’s anemiato chromosome breakage by DNA cross-linking agents. Cancer Res 33:1829–1836.

Seyschab H, Friedl R, Sun Y, Schindler D, Hoehn H, Schroeder-Kurth T(1995): Comparative evaluation of diepoxybutane sensitivity and cellcycle blockage in the diagnosis of Fanconi anemia. Blood 85:2233–2237.

Hans Joenje*Fre ArwertMei Lan KweeKamlesh MadanDepartments of Human Genetics and

Clinical GeneticsMedical Centre Free UniversityAmsterdam, The Netherlands

Holger HoehnInstitut fur HumangenetikUniversity of WurzburgWurzburg, Germany

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