Abstract
Approximately 25% of patients with Fanconi anemia (FA) have evidence of spontaneously occurring mosaicism as manifest by the presence of two sub-populations of lymphocytes, one of which is hypersensitive to cross-linking agents (e.g. mitomycin C) while the other behaves normally in response to these agents. The molecular basis of this phenotypic reversion has not yet been determined. We have investigated 8 FA patients with evidence of mosaicism. Epstein-Barr virus-immortalized lymphoblastoid cell lines established from these patients exhibited an IC50 for mitomycin C of 25 to >100 nM compared to a mean of 2 ± 2 nM for 20 nonmosaic FA patients and 49 ±11 nM for 8 healthy controls. In 3 patients who were compound heterozygotes for pathogenic FAC gene mutations the molecular mechanism of the mosaicism was investigated by haplotype analysis. The results indicated that an intragenic mitotic recombination must have occurred leading to a segregation of a wild-type allele in the reverted cells and suggested two patterns of recombination. In 1 patient a single intragenic crossover between the maternally and paternally inherited mutations occurred associated with markers located distally to the FAC gene; in the other 2 patients (sibs) the mechanism appears to have been gene conversion resulting in segregants which have lost one pathogenic mutation. In 6 of the 8 patients the hematological symptoms were relatively mild despite an age range of 9–30 years.
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Introduction
Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure and a diversity of somatic abnormalities including hyperpig-mentation of the skin (café au lait spots), growth delay, skeletal and renal anomalies, and hypogonadism [1–3]. FA cells exhibit increased spontaneous chromosomal instability and hypersensitivity to chromosomal breakage by cross-linking agents such as diepoxybutane (DEB) and mitomycin C (MMC). There are at least 5 FA complementation groups [4], group A being the most prevalent [5, 6]. The gene for group C has been cloned by expression cloning methodology [7] and mutations in this gene are responsible for an estimated 8% of FA cases worldwide [8] and at least 80% of cases with Ashkenazi Jewish ancestry [9, 10].
Hematopoietic failure is the major life-threatening complication in FA patients, which typically starts between 5 and 10 years of age. The aplastic phase may be followed by a myelodysplastic syndrome and acute myeloid leukemia [1, 11], which is usually fatal. Bone marrow failure may be cured by allogeneic bone marrow or umbilical cord blood transplantation [12, 13]. For FA-C patients without a suitable donor, gene therapy is being explored as a curative option [2, 14].
Hypersensitivity to cross-linking agents, as assessed in fresh phytohemagglutinin (PHA)-stimulated lymphocyte cultures, is currently the best criterion for the diagnosis of FA. Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines derived from FA peripheral blood samples usually are also hypersensitive to cross-linking agents, typically exhibiting 10- to 100-fold reduced half-maximal inhibitory concentrations (IC50 values) in standard growth inhibition tests compared to cell lines derived from healthy controls. However, over the last few years, it was found that EBV-immortalized lymphoblast lines established from approximately 25% of the FA patients in the EUFAR Cell Repository were MMC-resistant, in spite of the chromosomal breakage test on fresh lymphocytes being positive for DEB or MMC sensitivity. Such resistant cell lines are likely to derive from a subpopulation of B lymphocytes whose disease phenotype has reverted to the wild-type. Indeed, in some of these patients two sub-populations of T lymphocytes were clearly distinguishable in PHA-stimulated peripheral blood cultures, one exhibiting a typical FA-like response to MMC and one behaving like non-FA cells. A particularly well documented case of mosaicism has been reported previously in an FA patient whose lymphocyte cultures revealed a proportion of approximately 60% to be resistant to the chromosome breaking effect of MMC [15]. The possible complications due to the phenomenon of mosaicism for the diagnosis of FA patients have been discussed previously [15–17].
Here we document 8 mosaic FA cases, 2 of whom are brothers, whose EBV-immortalized lymphoblasts were MMC-resistant. Five patients were of an unknown complementation group, while 3 patients were deduced to belong to group C on the basis of mutations found in the FAC gene. In the FA-C cases we were able to establish the cause of the reversion by molecular genetic analysis of the FAC gene. Our results suggest that (1) reversion of the FA phenotype can occur spontaneously in hematopoietic stem or progenitor cells of FA patients, which may lead to sustained mosaicism of their hematopoietic system, (2) a single reverted stem cell may have the capacity to gradually replace affected progenitor cells leading to proficient clonal hematopoiesis, (3) in compound heterozygous patients phenotypic reversion may be caused by intragenic mitotic recombination generating a wild-type allele at the disease locus; however, additional mechanism(s) are likely to exist.
Materials and Methods
Patients
FA patients were selected from the EUFAR Cell Repository based on relative resistance to MMC of EBV-immortalized lymphoblast lines. Five patients (EUFA192, FA-1, EUFA806, EUFA449, and EUFA450) have been reported on before [15, 16, 18] (EUFA806 was ST in ref. 16). Because of the MMC resistance of the lymphoblast lines the patients could not be classified by functional complementation studies [4]; however, patients EUFA806, EUFA449 and EUFA450 could be assigned to complementation group C on the basis of pathogenic mutations found in the FAC gene. Patient EUFA806 was a compound heterozygote for 322delG (exon 1) and 1806insA (exon 14) [10, 19], whereas patients EUFA449 and EUFA450 (sibs) were compound heterozygotes for 322delG and the exon 14 mutation L554P [7, 18]. Clinical data of the patients are summarized in table 1.
Culture Methods and Chromosomal Breakage Assays
Diagnostic chromosomal breakage testing using PHA-stimulated fresh T lymphocyte cultures was as described previously [15]. Lymphoblastoid cell lines were established by immortalization of B lymphocytes with EBV according to a standard method [21] and cultured as described [4].
Generation of Chinese Hamster Hybrids
Primary diploid fibroblasts were obtained following standard procedures. Somatic cell hybrids from EUFA806 fibroblasts or lymphoblasts fused with Chinese hamster cells were obtained as described previously [22].
Mutation Detection
PCR of exons 1 and 14 of the FAC gene was performed as previously described [23]. PCR products of 5 independent amplifications were cloned in pBluescript and 10 randomly picked colonies were sequenced.
Haplotype Analysis
Microsatellite markers were amplified according to standard procedures. PCR was performed in a 15-µl volume containing 100 ng DNA, 0.2 mM dATP/dTTP/dGTP, 0.05 mM dCTP, 0.02 µl 32PdCTP (10 µCi/µl), 10 mM Tris HCl pH 9.0, 50 mM KCl, 0.01 % (w/v) gelatin, 0.1% Triton X-100, 1.5 mMMgCl2, and 0.2 units SuperTaq (HT Biotechnology LTD); the thermal profile was (30 cycles) 30 s 94°C, 60 s 56°C, and 30 s 72°C. The amplified fragments were separated on a 6% Polyacrylamide 7 M urea gel and visualized by autoradiography.
Assay of Clonality of Hematopoiesis
Clonality of hematopoiesis was assessed by PCR-based amplification of a polymorphic CAG repeat located on the X chromosome near methylation-sensitive restriction enzyme sites [24]. By comparing the PCR products obtained with and without digestion prior to the amplification the presence of active versus inactive X chromosomes was determined.
Results
Detection of Mosaicism in Lymphocyte Cultures
The experimental approach to demonstrate mosaicism in fresh peripheral blood lymphocyte cultures from FA patients has been described in a previous publication [15], where mosaicism was readily detected in patient FA-1 (at age 22) by assessing chromosomal breakage levels as induced by DEB (10–150 ng/ml), MMC (5–100 ng/ml) or cisplatin (0.2–1.5 mg/ml). At the highest clastogen concentrations the great majority of the healthy control cells were undamaged, while virtually 100% of the cells from a nonmosaic patient had chromosomal damage. FA-1 cells, on the other hand, had 62–69% undamaged cells, depending on the clastogen used. Using this methodology the level of mosaicism in T lymphocytes may be estimated, as summarized for the present patients in table 2.
Patients
The 8 patients that yielded a MMC-resistant lymphoblastoid cell line are listed in tables 1 and 2. The MMC resistance of the cell lines is indicated by the IC50 values (table 2), which were significantly elevated compared to the cell lines derived from non-mosaic cases. The level of mosaicism as estimated from chromosomal breakage rates in MMC-challenged lymphocyte cultures (see previous section) varied from patient to patient, from no detectable mosaicism (patient EUFA450) to apparently 100% MMC resistance (patient EUFA806); no data are available on patient EUFA554.
Changes in Mosaicism over Time
In 2 patients, EUFA192 and FA-1, it was possible to study the extent of mosaicism on different occasions over a relatively long period of time.
Patient EUFA192. This patient, who was previously reported as case FA-2 [15], was investigated for MMC-induced chromosomal breakage and diagnosed as FA only 1 month after birth and, when reinvestigated at 3 years of age, exhibited no sign of mosaicism [15]. Thirteen years later we established a lymphoblast line from this patient for the purpose of complementation analysis; however, the cell line was MMC-resistant and could therefore not be used for complementation testing. Since the MMC resistance suggested that the patient might have become mosaic, we repeated the chromosomal breakage test in peripheral blood T lymphocytes. Figure 1 illustrates that at 3 years of age chromosomal breakage was observed in virtually all cells after a 3-day treatment with MMC at 100 ng/ml, whereas at age 16 only a small minority (> 10%) of the cells exhibited FA-like high breakage levels, the remaining cells responding as the healthy control. This confirmed that the patient had indeed acquired phenotypically reverted cells, to the extent that almost all MMC-sensitive T lymphocytes were replaced by MMC-resistant ones. To explain this phenomenon we assumed that a pluripotent stem cell in the patient’s bone marrow had undergone a stable phenotypic reversion, after which its progenitor cells, because of their proliferative advantage [25, 26], had gradually replaced the affected progenitor cells. Since reversion is supposed to be a rare event that is not likely to occur independently in multiple stem cells, this explanation would predict that the patient’s hematopoiesis should have become clonal, or be at least clearly skewed towards clonality. As demonstrated by a PCR-based assay of polymorphic X chromosome markers [24], clonal hematopoiesis in the patient was confirmed. In contrast, fibroblasts cultured from a skin biopsy of the patient were polyclonal (fig. 2).
Patient FA-1. This patient was reported previously [15] as a clear example of mosaicism, with 69% of his T lymphocytes behaving like healthy control cells. During a 5 year follow-up period, to monitor the proportion of reverted cells, no significant change was observed (table 2), indicating that in this patient within this period of time an equilibrium had been reached. Given the relatively long period of time the mosaic status had existed in this patient, we may assume that also in this patient at least one pluripotent stem cell must have undergone stable phenotypic reversion.
Genetic Basis of Phenotypic Reversion in FA-C Patients EUFA806, EUFA449 and EUFA450
Patient EUFA806. This patient was described previously [patient ST in 16] as an example of a mosaic FA patient with practically 100% of her lymphocytes behaving like normal control cells. Classification of this patient as FA-C was based on finding mutations in the FAC gene. The patient was found to be a compound heterozygote for the exon 1 mutation 322delG [7, 10] and the exon 14 mutation 1806insA [19]. Whereas fibroblasts from the patient were sensitive to MMC, an EBV-immortalized lymphoblastoid cell line derived from the patient was MMC-resistant (table 2); the same level of resistance was observed with DEB and cisplatin. Both mutations were detected in the fibroblasts as well as in the MMC-resistant lymphoblasts. A mitotic crossover between the mutated sites in compound heterozygotes was considered a plausible mechanism to explain the occurrence of phenotypically reverted FA cells (fig. 3). If an intragenic mitotic recombination event between the two FAC mutations was responsible for the phenotypic reversion, a wild-type allele should exist in the patient’s lymphoblasts. To examine this possibility the two FAC alleles had to be studied separately. To this end, we fused the patient’s sensitive fibroblasts as well as her resistant lymphoblasts with Chinese hamster cells to produce hybrids, which, due to random chromosome loss, contained single copies of the human chromosome 9. Fusions involving the patient’s fibroblasts yielded 20 clones, which were investigated for the presence of the human chromosome 9 by microsatellite typing. Ten of the hybrid clones had retained both copies of chromosome 9, the remaining 10 having only one copy. Remarkably, all of the latter clones apparently had retained the chromosome carrying the 322delG mutation, indicating preferential loss of the chromosome 9 having the 1806insA mutation. Nevertheless, the results are consistent with the two mutations in the fibroblasts residing on different chromosomes. Fusions of the patient’s MMC-resistant lymphoblasts with a Chinese hamster cell line resulted in 23 hybrid clones containing two chromosomes 9 and only one clone with a single human chromosome 9. This chromosome, which contained neither the 322delG nor the 1806insA mutation, apparently carried a wild-type FAC allele.
If the wild-type allele had indeed originated from a single mitotic crossover, a switch of haplotypes was to be expected at the disease locus. Haplotype analysis using polymorphic CA-repeat markers flanking the FAC locus was carried out in the patient’s fibroblasts, her MMC-resistant lymphoblasts and the fusion hybrids containing a single chromosome 9. Comparison of the pattern found in fibroblasts with that in the fibroblast/hamster fusion hybrid established which haplotype was associated with each of the two mutations (fig. 4). In the lymphoblast/hamster hybrid the wild-type allele apparently had parts of both haplotypes, i.e. the proximal part of the 322delG haplotype (including markers on the p-arm) and the distal part of the 1806insA haplotype. This result indicated that an intragenic recombination event had generated a wild-type allele in the MMC-resistant lymphoblastoid cell line. These results also permitted the deduction that the 3’ end of the FAC gene (containing the 1806insA mutation) must be oriented towards the centromere of chromosome 9 (fig. 5).
Since heterozygous carriers of a FA mutation have a normal (non-FA-like) response to cross-linkers, the intragenic recombination event deduced to have occurred in the patient’s hematopoietic system and leading to the production of a wild-type allele appears to provide an adequate explanation for the observed phenotypic reversion to MMC resistance.
Patients EUFA449 and EUFA450. These patients, who are siblings, are compound heterozygotes for mutations in exons 1 and 14, that is 322delG and L554P [7, 10, 18], respectively. These mutations were found in genomic DNA isolated from the patients’ peripheral blood samples. EBV-immortalized lymphoblastoid cell lines established from the patients were both found to be MMC resistant. Screening for the mutations in the lymphoblasts revealed that in both cell lines the exon 1 mutation 322delG was not present. If, as in case EUFA806, a single intragenic crossover had been responsible for this result, loss of heterozygosity would be expected for polymorphic markers distal to the gene (fig. 3). However, all distal markers investigated had retained heterozygosity [results not shown]. Therefore, the recombination mechanism of gene conversion has most likely been responsible for the loss of the 322delG mutation [27].
Discussion
Possible Causes of Mosaicism
Theoretically, mosaicism in FA patients might originate from genetic changes at the disease locus or at modifier genes capable of alleviating the disease phenotype. With respect to changes at the disease locus, two types of event may be distinguished. First, during development, an individual cell of a heterozygous carrier of an FA gene mutation may acquire a pathogenic mutation in the second allele causing all descendants from this cell to be affected by FA. Depending on the age at which this happens it will affect many or only a minor proportion of the individual’s cells. This may be called ‘forward mosaicism’. The second possibility may occur in a homozygously affected patient in whom one or more cells reverse to a heterozygous carrier state by the generation of a wild-type allele at the disease locus (‘reverse mosaicism’). Where the FA gene mutation(s) are unknown in a patient, it is not currently possible to determine how the mosaicism originated. Clearly, as shown by our data, in the 3 patients who were found to belong to group FA-C by mutation screening (EUFA806, EUFA449 and EUFA450) the generation of a wild-type allele at the disease locus is most likely to have been responsible for their mosaicism. Forward mosaicism is a formal possibility in the unclassified patients EUFA192 and EUFA631, since these patients were singleton cases and there are no data to confirm that they inherited two mutated FA alleles from their parents. However, all other cases were from multiplex families, which strongly suggests that they originated from homozygously affected zygotes. Ultimate proof for ‘reverse mosaicism’ in these cases, however, requires the determination of their complementation group, finding mutations in the corresponding gene and confirming the presence of these mutations in the parents.
Detection of Mosaicism in FA Patients
Hypersensitivity to chromosomal breakage by cross-linking agents is the essential criterion to confirm the diagnosis in a patient suspected of having FA [28]. This test is normally carried out with 72 h PHA-stimulated peripheral blood cultures, incubated with and without a cross-linking agent, like DEB or MMC, which are added at a concentration that evokes a significant clastogenic response in FA but not in non-FA individuals. This test readily detects clastogen-resistant cells, when occurring at relatively high levels [15], but is not suitable for detecting low levels of mosaicism (e.g. 20% or less). However, EBV-immortalized cell lines established from low-level mosaic patients may still reveal MMC resistance. Since FA lymphocytes are more difficult to immortalize than non-FA lymphocytes, lymphoblastoid cell lines established from mosaic patients will preferentially derive from the sub-population of reverted cells, even if reverted cells occur as a minority, i.e. not detectable by chromosomal breakage analysis (e.g. patient EUFA450). In view of the clinical implications that may be associated with the phenomenon of mosaicism in FA, it may be advisable to establish EBV-immortalized lymphoblastoid cell lines from all FA patients routinely, and to test their level of cross-linker sensitivity for possible low-level mosaicism.
Mechanisms of Phenotypic Reversion
Mosaicism due to mitotic recombination has been described in Bloom syndrome (BS), both in vitro [29] and in vivo [30]. Lymphocytes from BS patients typically exhibit much increased levels of sister chromatid exchanges. As in FA, about 20% of BS patients appear to be mosaics, in particular those whose parents do not share a common ancestor and therefore are more likely to be compound heterozygotes. In such patients a minority of lymphocytes are detected with a normal level of sister chromatid exchanges. Ellis et al. [30] found that in 5 out of 11 such mosaic patients loss of heterozygosity had occurred for polymorphic microsatellite markers distal to the gene BLM, which is consistent with an expected 50% of this type of segregant (fig. 3B), assuming that recombination occurs after the DNA has been replicated (see below).
The FA-C patients reported here illustrate that intragenic mitotic recombination can also be a cause of mosaicism in compound heterozygote FA patients. Two types of recombination could be inferred from the genotypes of the reverted cells: (1) a single intragenic crossover between the maternally and paternally inherited mutations, associated with haplotype switching for markers located distally to the disease gene (patient EUFA806), and (2) gene conversion, leading to segregants having lost one pathogenic mutation (patients EUFA449 and EUFA450). Both types of recombination are compatible with their occurrence after the DNA having been replicated, as postulated by Ellis et al. [30] for BS; however, they are also compatible with the recombination having taken place in prereplicative DNA (fig. 3A).
The inferred recombination mechanisms require a compound heterozygous genotype in the patients. Ellis et al. [30] found 3 cases with mosaicism among 68 (4%) of their BS patients who were judged to be homozygous by descent at the BLM locus, implying that a mechanism other than intragenic somatic recombination must have been responsible for mosaicism in these BS cases. Such cases may represent the involvement of modifier genes that, when altered in a specific way, may be capable of alleviating the cellular disease phenotype. Patient EUFA554, who was born in a consanguineous multiplex family, might be an analogous case in FA. Thus, also in FA mechanisms other than intragenic mitotic recombination are likely to exist that can generate phenotypically reverted cells.
Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency was recently reported [31]; however, the molecular mechanism underlying this reversion has not been established.
Likelihood of Reversion and Level of Mosaicism
The phenomenon of phenotypic reversion has also been observed in ataxia telangiectasia (AT), where lymphoblastoid cell lines from some patients appear to have lost their AT phenotype [32]. FA, BS and AT share spontaneous chromosomal instability as a cellular feature, which in AT is associated with a phenotype of hyperrecombination [33]. In FA an additional phenomenon might explain the apparently high proportion of reverted cells in some patients: hematopoietic progenitor cells originating from a reverted stem cell are likely to have a proliferative advantage over poorly proliferating homozygous FA progenitors [25, 26], leading to progressive replacement of the affected progenitor cells. Clonal hematopoiesis in patient EUFA192 and the hematological condition of this patient suggest that MMC-resistant cells are derived from a single reverted pluripotent stem cell and that the probability of reversion in stem cells may be quite low. The discordant course of the disease in the monozygous twin patients EUFA53 (mosaic; no hematological symptoms) and EUFA54 (severe symptoms; transplanted; however, no information available on mosaicism) again suggests a low probability for mosaicism to occur. On the other hand, in the siblings EUFA449 and EUFA450 the same type of genetic reversion (gene conversion) occurred independently, suggesting that the probability of reversion cannot be exceedingly low.
Course of the Disease in Mosaic Patients
The severity of the disease phenotype in FA-C patients has been correlated with the type of mutation, the IVS4+4A→T mutation being associated with a severe and the 322delG mutation with a relatively mild course of the disease [10, 34]. A possible correlation between course of hematological disease and (level of) mosaicism has not been studied systematically. Our limited data suggest a negative correlation between level of mosaicism and severity of hematological symptoms. Most of the patients with clearly demonstrable levels of MMC-resistant T lymphocytes (table 2) had no or only very mild hematological symptoms, in spite of an age range of 9–30 years [table 1; cf.ref. 1].
Clinical Implications
Our data suggest that mosaicism might be associated with a relatively mild hematological course, even though more research would be necessary to substantiate this point. Below, we discuss some considerations that might be relevant for the clinical management of mosaic FA patients in relation to bone marrow transplantation (BMT), gene therapy and leukemia. BMT of FA patients with HLA-identical donors has been increasingly successful after the original conditioning treatment developed for non-FA patients had been attenuated to meet the increased sensitivity of FA patients to cyclophosphamide [12]. In spite of this, BMT fails in over 30% of such transplants. It is conceivable that mosaic patients would do less well in BMT than nonmosaics, since the attenuated ablation protocol may fail to eliminate the phenotypically reverted cells, which may subsequently compromise engraftment.
In FA-C patients undergoing gene therapy, CD34+cells are transduced ex vivo with a wild-type FAC gene-expressing retroviral vector. Reinfusion into the patient is intended to generate a mosaic bone marrow in which corrected stem cells initially coexist with affected stem cells, but are expected to gradually take over hematopoiesis. Such an approach would, however, create little benefit for a mosaic patient whose bone marrow has already undergone the desired change spontaneously. Clonal hematopoiesis in patient EUFA192 suggests that spontaneous or gene therapeutic correction of a single pluripotent stem cell may be sufficient to restore proficient hematopoiesis.
Implications for Leukemia
FA patients are prone to develop cancer, in particular acute myeloid leukemia, presumably as a result of the chromosomal instability inherent in the disease phenotype [1–3, 35]. Leukemia in FA patients is extremely difficult to manage clinically and is usually fatal. The presence of phenotypically reverted cells in mosaic patients makes this situation even more complicated. The use of cross-linking agents (e.g. cyclophosphamide, cisplatin) for chemotherapeutic treatment should be carefully considered, as such agents will differentially affect the survival of the FA and non-FA cells present in a mosaic patient.
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Acknowledgements
We thank Franca di Summa and Carola van Berkel for expert technical assistance, Prof. Dr. Leo P. ten Kate for valuable comments on the manuscript, and Dr. Vicky A. Athens for her cooperation in this study. This work was supported by the European Cancer Centre Amsterdam (ECC), EUFAR (a concerted action sponsored by the European Union, contract PL931562), the FA Research Fund, Inc., Eugene, Oreg., The Medical Research Council (UK), and the patient support organizations in Italy, France, Germany and The Netherlands.
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Ten Foe, J.R.L., Kwee, M.L., Rooimans, M.A. et al. Somatic Mosaicism in Fanconi Anemia: Molecular Basis and Clinical Significance. Eur J Hum Genet 5, 137–148 (1997). https://doi.org/10.1007/BF03405891
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DOI: https://doi.org/10.1007/BF03405891