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Keywords:

  • bone marrow failure;
  • Fanconi anaemia;
  • genetics;
  • mutations;
  • clinical aspects

Summary

  1. Top of page
  2. Summary
  3. Patient and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Fanconi anaemia (FA) is a genetically heterogeneous chromosome instability syndrome characterised by bone marrow failure and congenital anomalies. Although an increasing number of reports suggest that reversion mosaicism noted in peripheral blood lymphocytes (PBLs) is associated with mild haematopoietic failure in FA, myeloid cells are rarely directly examined. We here report a patient with prolonged mild pancytopenia in whom proliferation of revertant cells was detected in mature myeloid cells but not in PBLs. While this patient had inherited heterozygous mutations, 2546delC and 3720–3724del, in the major FA gene FANCA, Epstein–Barr virus-immortalised lymphoblastoid cells from the patient had 2546C > T instead of 2546delC, resulting in expression of a functional missense protein. As the identical reversion was detected in polymorphonuclear granulocytes and mononuclear phagocytes, sustained haematopoiesis in the patient can be attributed to a selective growth advantage of revertant myeloid cells. It is noteworthy that such a myeloid lineage-selective mosaicism is overlooked in routine examination of PBLs. Recognition of this status will expand the role of reversion mosaicism in the pathophysiology of FA.

Fanconi anaemia (FA) is an inherited chromosome instability syndrome characterised by congenital anomalies, progressive bone marrow failure, leukaemia susceptibility and cellular hypersensitivity to DNA crosslinkers, such as mitomycin C (MMC) (Tischkowitz & Dokal, 2004). FA has at least 12 different complementation groups (FA-A, B, C, D1, D2, E, F, G, I, J, L and M) and 11 FA genes have been identified (FANCA, FANCB, FANCC, FANCD1 /BRCA2, FANCD2, FANCE, FANCF, FANCG /XRCC9, FANCJ /BRIP1, FANCL, /PHF9 and FANCM) (Meetei et al, 2004; Tischkowitz & Dokal, 2004; Levitus et al, 2005; Levran et al, 2005; Meetei et al, 2005). These products function in a common pathway, the FA pathway, wherein nuclear multiprotein complex FANCA/B/C/E/F/G/L/M-dependent monoubiquitination of FANCD2 plays a critical role in DNA damage response (Garcia-Higuera et al, 2001).

Fanconi anaemia patients characteristically show a broad range of clinical phenotypes. Some patients have multiple severe anomalies including organ defects, whereas others have minor physical abnormalities such as skin pigmentation. In addition, onset and progression of haematological disorders, bone marrow failure, myelodysplasia and leukaemia, are variable (Faivre et al, 2000; Kutler et al, 2003; Rosenberg et al, 2004). The phenotypic diversity is partly attributed to the genetic heterogeneity and mutation types (Gillio et al, 1997; Faivre et al, 2000; Wagner et al, 2004). Recently, increasing attention has been focused on the role of somatic mosaicism in the variability of haematological symptoms (Lo Ten Foe et al, 1997; Waisfisz et al, 1999; Gregory et al, 2001; Gross et al, 2002; Soulier et al, 2005). Clonal expansion of a haematopoietic stem cell with reversion can restore normal haematopoiesis. However, in most previous mosaic cases, reversion was noted by appearance of peripheral blood lymphocytes (PBLs) with normal sensitivity to DNA crosslinkers but, to our knowledge, myeloid cells were directly examined in only a single case, (Gregory et al, 2001). We report a patient with persistent mild pancytopenia in whom a genetic reversion was detected in mature myeloid cells but not in PBLs. We suggest that myeloid lineage-selective mosaicism may partly account for the variability of haematological disorders in FA.

Patient and methods

  1. Top of page
  2. Summary
  3. Patient and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patient

A 22-year-old Japanese female (FA67) was diagnosed with FA at 4 years of age, based on mild pancytopenia (white blood cells 2·5 × 109/l; haemoglobin 11·6 g/dl; platelet count 70 × 109/l), congenital anomalies (microcephaly, micrognathia and skin pigmentation) and a positive chromosome breakage test. Medical records show that >80% of PBLs showed chromosome breaks under treatment with MMC. Her blood cell counts had been stable for the last 18 years (Fig 1). Total leukocyte and absolute neutrophil counts had remained around 2·0 × 109/l and 1·0 × 109/l. Platelet counts slowly increased between the ages of 14 and 18 years, and then stabilised at around 100 × 109/l or higher. Haemoglobin levels gradually decreased but recovered to approximately 10 g/dl after recent iron supplementation. A bone marrow sample obtained when the patient was aged 19 years was slightly hypoplastic; cytogenetic analysis of the bone marrow revealed karyotype 46, XY, add(1)(p10) in four of 20 mitotic cells analysed. She received no blood transfusion or treatment with glucocorticoid or androgen and had no history of pregnancy.

image

Figure 1. Time course of haematological parameters. Blood cell count curves from FA67 are shown over 18 years after diagnosis at the age of 4. Total white blood cell counts (WBC) and absolute neutrophil counts (ANC) are shown in the upper panel, and haemoglobin levels (Hb) and platelet counts (Plt) are shown in the lower panel. Iron (Fe) was orally administered for iron deficiency anaemia.

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Separation and cultures of blood cells

Blood samples were obtained from subjects after their informed consent and with the approval of the institutional review board. Mononuclear cells and polymorphonuclear granulocytes (approximately 90% purity) were isolated from peripheral blood by density gradient centrifugation using PolymorphprepTM (Axis-Shield PoC AS, Oslo, Norway). Mononuclear cells depleted of phagocytic cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) with phytohaemagglutinin for 96 h and used as PBLs for further analyses or infected with Epstein–Barr virus to generate a lymphoblastoid cell line (LCL). The LCL was cultured in RPMI1640 medium containing 10% FBS.

DNA sequencing

Genomic DNA was extracted from indicated cells using standard procedures. Exons of FANCA were amplified and sequenced on an ABI 3100 automated sequencer (Applied Biosystems, Foster City, CA, USA) as described previously (Yagasaki et al, 2004). Polymerase chain reaction (PCR)-amplified fragments of exon 27 from genomic DNA of indicated cells were subcloned and sequenced.

Protein analyses

Levels of FANCA and monoubiquitinated and non-ubiquitinated forms of FANCD2 were determined using immunoblotting analyses of whole cell extracts with anti-FANCA and anti-FANCD2 antibodies as described previously (Adachi et al, 2002; Yagasaki et al, 2004).

Generation of transformants of GM6914 fibroblasts

SV40-immortalised FANCA-null human fibroblasts GM6914 were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. GM6914 cells were stably transduced with cDNAs of mutant FANCA generated by site-directed mutagenesis or wild-type (Wt) FANCA as described previously (Adachi et al, 2002; Yagasaki et al, 2004).

Cellular sensitivities to MMC

Cellular sensitivities of LCL and GM6914 transformants were determined using a cell survival assay as described previously (Adachi et al, 2002; Yagasaki et al, 2004).

Results

  1. Top of page
  2. Summary
  3. Patient and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cultured PBLs from FA67 exhibited a marked G2 arrest (data not shown), a cellular phenotype characteristic of FA, and lacked in FANCA protein expression and FANCD2 monoubiquitination (Fig 2A, lane 3). These data suggest defective activation of the FA pathway as a result of biallelic null mutations of FANCA (Shimamura et al, 2002). By contrast, cultured LCL from the patient demonstrated restored FANCA protein expression and FANCD2 monoubiquitination (Fig 2A, lane 6), suggesting genetic reversion in these cells (Soulier et al, 2005). Consistently, these cells showed normal MMC sensitivity in the cytotoxicity assay (Fig 2B).

image

Figure 2. Lack of FANCA protein expression in the peripheral blood lymphocytes (PBL)s and its restoration in the lymphoblastoid cell line (LCL). (A) FANCA protein expression and FANCD2 monoubiquitination in PBLs and LCLs from FA67, a FANCA-null patient [A(−)] and a normal control [A(+)]. Cell lysates from these cells were immunoblotted with anti-FANCA and anti-FANCD2 antibodies. Protein loading was assessed by probing with anti-tubulin β antibody. Protein bands corresponding to FANCD2 (D2) and a monoubiquitinated form of FANCD2 (Ub-D2) are indicated by arrowheads. (B) Mitomycin C (MMC) sensitivities of LCLs from FA67, a FANCA-null patient [A(−)] and a normal control [A(+)] were determined in the cytotoxicity assay.

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In order to determine the genetic basis of the discrepancy between the PBLs and LCL from FA67, we sequenced genomic DNA from these cells. Heterozygous mutations of FANCA, 2546delC (Fig 3, top) and 3720–3724del (not shown), were identified in the PBLs. These mutations were detected in buccal cells and proved to be inherited from the patient's parents (Table I). On the contrary, genomic DNA from the LCL had 2546C > T (Fig 3, middle), instead of 2546delC, with 3720–3724del. Thus, functional correction of the LCL can be attributed to expression of a missense FANCA (Ser848Phe) protein encoded by the altered allele (2546C > T). To confirm this notion, we assessed functions of the missense (Ser848Phe) FANCA protein. For this purpose, we stably expressed the mutant and Wt proteins in GM6914 FANCA-deficient fibroblasts at similar levels (Fig 4A). FANCA (Ser848Phe) restored FANCD2 monoubiquitination either at a basal state or after treatment with MMC (Fig 4B) and corrected cellular sensitivity to MMC in the cell survival assay (Fig 4C), similarly to Wt FANCA.

image

Figure 3. DNA sequencing of FANCA in the peripheral blood lymphocytes (PBL)s, lymphoblastoid cell line (LCL) and granulocytes. 2546delC in the PBLs and 2546C > T in the LCL and granulocytes from FA67 are indicated by arrowheads.

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Table I.  Mutations in genomic DNA from various cells of FA67 and PBLs of her parents.
 FatherMotherFA67
PBLsPBLsBuccal cellsPBLsLCLGranulocytes
  1. PBLs, peripheral blood lymphocytes; LCL, lymphoblastoid cell line.

Allele 13720–3724delWild-type3720–3724del3720–3724del3720–3724del3720–3724del
Allele 2wild-type2546delC2546delC2546delC2546C > T2546C > T
image

Figure 4. Functional expression of the FANCA mutant protein (Ser848Phe). (A) Cell lysates from GM6914 cells stably expressing wild-type (Wt) and mutant (Ser848Phe) FANCA proteins and mock-transfected control cells were immunoblotted with anti-FANCA and anti-tubulin β antibodies. (B) The same transformants were cultured in the absence (−) or presence (+) of 40 ng/ml MMC for 18 h. Cell lysates were immunoblotted with anti-FANCD2 antibody. Protein bands corresponding to FANCD2 (D2) and a monoubiquitinated form of FANCD2(Ub-D2) are indicated by arrows. (C) MMC sensitivities of the GM6914 transformants expressing Wt and mutant (Ser848Phe) FANCA proteins and mock-transfected control cells.

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Notably, the above biochemical and genetic disagreements between the PBLs and LCL were reproduced in two independent blood samples from FA67. These observations led us to the hypothesis that proliferation of haematopoietic cells with the reverted allele (2546C > T) may account for prolonged mild pancytopenia. To test this, we sequenced the FANCA gene using genomic DNA from polymorphonuclear granulocytes and mononuclear phagocytes and we found that both cells carried the identical reversion (Fig 3, bottom and data not shown). To further confirm the genetic difference between granulocytes and the PBLs, we subcloned and sequenced PCR-amplified exon 27 fragments from these two populations. The reverted allele (2546C > T) was predominant in granulocytes but undetectable in the PBLs (Table II). These results suggested that growth advantage of revertant cells in the myeloid-lineage accounted for the mild neutropenia in FA67. Although the predominance of revertant cells in the LCL may result from in vitro selection during immortalisation, it indicated the presence of revertant B cells. This meant that the reversion was not restricted to myelomonocytic precursors, thus mild anaemia and thrombocytopenia in FA67 can probably be attributed to in vivo proliferation of revertant cells among erythroid and megakaryocytic cells.

Table II.  Analysis of subclones of exon 27 in peripheral blood lymphocytes (PBL)s and granulocytes.
 Wild-type2546delC2546C > T
PBLs11/2413/240/24
Granulocytes10/191/198/19

Discussion

  1. Top of page
  2. Summary
  3. Patient and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A critical determinant for the haematological consequences of reversion mosaicism in FA is the lineage in which the reversion occurs and revertant cells proliferate. The association of reversion mosaicism in PBLs with mild pancytopenia is considered to result from the occurrence of reversion in a lympho-myeloid stem cell and its proliferation in both myeloid and lymphoid lineages (Lo Ten Foe et al, 1997; Waisfisz et al, 1999; Gregory et al, 2001; Gross et al, 2002; Soulier et al, 2005). While this was shown by analyses of haematopoietic cell colonies of various lineages in a single patient (Gregory et al, 2001), myeloid cells have not been examined in other cases. The present case suggests that examination of mature myeloid cells provides useful information to correlate a genetic reversion with haematological data and should be considered even when reversion mosaicism in PBLs has not been detected.

In general, two main mechanisms are possible for the lineage-selective proliferation of revertant cells. One is that somatic reversion occurs in a lineage-restricted progenitor cell. An alternative mechanism is that while reversion occurs in a multi-lineage common precursor cell, revertant cells have a growth advantage in a lineage-selective manner, because of the functional importance of the relevant protein in the lineage. The latter probably accounts for selective proliferation of revertant T cells in severe combined immunodeficiency disorders because of inherited mutations of the γc cytokine receptor and adenosine deaminase genes (Stephan et al, 1996; Ariga et al, 2001). This has been confirmed by recent data from a patient with Wiskott–Aldrich syndrome, in whom, despite the detection of a genetic reversion in both B and T cells, revertant cells proliferated selectively among T cells (Konno et al, 2004). It has not been determined why revertant cells proliferated in the myeloid-lineage but not in PBLs in FA67. However, as FA proteins seem to have more important functions in myeloid cells than in lymphoid cells from its clinical phenotypes, myeloid lineage-selective proliferation of revertant cells in FA can occur although it has not been reported so far. We therefore propose that FA67 is not a unique case.

Mutational studies of the FANCA gene in various ethnic groups have revealed that a wide variety of mutations, including nucleotide substitutions, small deletions/insertions and large deletions (Levran et al, 1997; Morgan et al, 1999; Tachibana et al, 1999; Tipping et al, 2001; Yagasaki et al, 2004). The FANCA gene is rich in direct repeats and homonucleotide tracts, which frequently cause small insertions and deletions partly through slipped-strand mispairing (Levran et al, 1997; Waisfisz et al, 1999). The poly(T) sequence surrounding 2546C (Fig 3) is likely to be susceptible to both forward and reverse mutations. As 2546delC is a prevalent (about 20%) mutant allele of FANCA in Japanese FA patients (Tachibana et al, 1999; Yagasaki et al, 2004), it is an interesting possibility that reversion at this site is recurrent in different individuals, as noted in families with Wiskott–Aldrich syndrome (Wada et al, 2003, 2004; ). Indeed, we recently detected the same reversion in PBLs of an unrelated patient (FA98) (data not shown).

In addition to our cases, in vivo genetic reversions have been documented in 11 FA patients (Table III). The present study of FA67 shows that routine examination of PBLs can miss myeloid lineage-selective reversion and when seeing patients with mild haematological symptoms and/or when patient-derived LCLs have reverted phenotypes without apparent reversion mosaicism in PBLs, we should keep in mind the possibility of selective growth of revertant myeloid cells. Although it is not easy to demonstrate genetic reversions in myeloid-lineage stem/progenitor cells, examination of mature myeloid cells is a practical and useful alternative method. Recognition of lineage-selective reversion mosaicism will provide new insights into the pathophysiology of FA.

Table III.  Summary of FA patients with in vivo genetic reversion.
Patient codeGeneInherited mutation[RIGHTWARDS ARROW]ReversionMechanism of reversionRevertant cellsEffect on phenotypeReferences
  1. FA, Fanconi anaemia; PBLs, peripheral blood lymphocytes; LCL, lymphoblastoid cell line; CBC, complete blood cell count; WBC, white blood cell count.

FA67FANCA2546delC[RIGHTWARDS ARROW]2546C > TSlippage?Granulocytes, mononuclear phagocytes, LCLMild pancytopeniaCurrent study
FA98FANCA2546delC[RIGHTWARDS ARROW]2546C > TSlippage?PBLsProgressive pancytopeniaCurrent study
EUFA704FANCA1615delG[RIGHTWARDS ARROW]1615delG + 1637delA + 1641delTSecond site deletionsPBLs, LCLWaisfisz et al, 1999
EUFA393FANCA3559insG[RIGHTWARDS ARROW]3559insG + 3580insCGCTGSecond site insertionsPBLs and LCLWaisfisz et al, 1999
IFAR557-2FANCA2815–2816ins19[RIGHTWARDS ARROW]Wild-typeBack mutationPBLs, LCL, haematopoietic stem cellsMild pancytopenia [RIGHTWARDS ARROW] progression/ clonal evolutionGregory et al, 2001
URDFANCA856C > T[RIGHTWARDS ARROW]Wild-typeBack mutationPBLs, LCLMild pancytopeniaGross et al, 2002
STTFANCA862G > T[RIGHTWARDS ARROW]Wild-typeBack mutationPBLs, LCLMild pancytopeniaGross et al, 2002
MRBFANCA971T > G[RIGHTWARDS ARROW]Wild-typeBack mutationPBLs, LCLMild pancytopeniaGross et al, 2002
EUFA173FANCA2852G > A[RIGHTWARDS ARROW]Wild-typeBack mutationPBLs, LCLMild pancytopeniaGross et al, 2002
EUFA506FANCC1749T > G[RIGHTWARDS ARROW]1748C > T + 1749T > GCompensatory missense mutationPBLs, LCL-Waisfisz et al, 1999
EUFA806FANCC67delG/1806insA[RIGHTWARDS ARROW]Wild-typeMitotic recombinationPBLs, LCLNormal CBCLo Ten Foe et al, 1997
EUFA449FANCC67delG[RIGHTWARDS ARROW]Wild-typeGene conversion?PBLs, LCLMild pancytopeniaLo Ten Foe et al, 1997
BRCA2/FANCD18732C > A[RIGHTWARDS ARROW]8731T > G + 8732C > ACompensatory missense mutationLeukaemic cellsResistance to DNA crosslinkersIkeda et al, 2003

Acknowledgements

  1. Top of page
  2. Summary
  3. Patient and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture of Japan and grants from the Ministry of Health, Labor and Welfare of Japan.

We thank Keiko Nakazato for her technical assistance.

References

  1. Top of page
  2. Summary
  3. Patient and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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