Genotype–phenotype correlations in Fanconi anemia

Mutation Research 668 (2009) 73–91

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Review

Genotype–phenotype correlations in Fanconi anemia Kornelia Neveling, Daniela Endt, Holger Hoehn, Detlev Schindler ∗ Department of Human and Medical Genetics, University of Wurzburg, Biozentrum, Am Hubland, D-97074 Wurzburg, Germany

a r t i c l e

i n f o

Article history: Received 24 September 2008 Received in revised form 30 March 2009 Accepted 12 May 2009 Available online 21 May 2009 Keywords: Fanconi anemia DNA repair Genotype–phenotype correlations Bone marrow failure Cancer susceptibility Stem cells Genome instability Mechanisms of ageing Accelerated ageing Segmental progeroid syndromes

a b s t r a c t Although still incomplete, we now have a remarkably detailed and nuanced picture of both phenotypic and genotypic components of the FA spectrum. Initially described as a combination of pancytopenia with a limited number of physical anomalies, it was later recognized that additional features were compatible with the FA phenotype, including a form without detectable malformations (Estren–Dameshek variant). The discovery of somatic mosaicism extended the boundaries of the FA phenotype to cases even without any overt hematological manifestations. This clinical heterogeneity was augmented by new conceptualizations. There was the realization of a constant risk for the development of myelodysplasia and certain malignancies, including acute myelogenous leukemia and squamous cell carcinoma, and there was the emergence of a distinctive cellular phenotype. A striking degree of genetic heterogeneity became apparent with the delineation of at least 12 complementation groups and the identification of their underlying genes. Although functional genetic insights have fostered the interpretation of many phenotypic features, surprisingly few stringent genotype–phenotype connections have emerged. In addition to myriad genetic alterations, less predictable influences are likely to modulate the FA phenotype, including modifier genes, environmental factors and chance effects. In reviewing the current status of genotype–phenotype correlations, we arrive at a unifying hypothesis to explain the remarkably wide range of FA phenotypes. Given the large body of evidence that genomic instability is a major underlying mechanism of accelerated ageing phenotypes, we propose that the numerous FA variants can be viewed as differential modulations and compression in time of intrinsic biological ageing. © 2009 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple genotypes and multiple clinical phenotypes connected by a singular cellular phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. FA cellular phenotype: chromosome breakage and chromosome aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. FA cellular phenotype: crosslinker sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. FA cellular phenotype: cell cycle disturbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. FA cellular phenotype: oxygen sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. FA cellular phenotype: deregulated apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. FA cellular phenotype: RAD51 foci formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genotype–phenotype correlations according to complementation group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Distinctive clinical phenotypes as function of complementation group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. With few exceptions, primacy of type of mutation over type of gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AA, aplastic anemia; AML, acute myelogenous leukemia; Ask1, apoptosis signal-regulating kinase 1; BIC, breast cancer information core; BMF, bone marrow failure; BRCA, breast cancer; BRCT, BRCA1 C-terminal; CAB scores, congenital abnormality scores; CALMs, café-au-lait macules; CDDP, cisplatin(um) or cisdiamminedichloridoplatinum(II); COFS syndrome, cerebro-oculo-facio-skeletal syndrome; DEB, diepoxybutane; ERCC1, excision-repair cross-complementing gene 1; EVI1, ecotropic virus integration site 1 gene; FA, Fanconi anemia; FANC , FA gene or protein of specified subtype; GFP, (enhanced) green-fluorescent protein; GVHD, graft-versus-host disease; H3P, phospho-histone H3; HDR, homology-directed DNA repair; HSCT, homologous stem cell transplantation; ICL, DNA-interstrand crosslink(ing); IFAR, International Fanconi Anemia Registry; kDa, kilodalton; LCL, B lymphocyte-derived EBV-transformed lymphoblastoid cell line; MCA, multiple congenital anomalies; MDS, myelodysplastic syndrome; MEFs, murine embryonic fibroblasts; MMC, mitomycin C; NBS, Nijmegen breakage syndrome; NF, neurofibromatosis; NM, nitrogen mustard; RAD, radiationsensitive; ROS, reactive oxygen species; SCC, squamous cell carcinoma; SCE, sister chromatid exchange; TNF, tumor necrosis factor; wt, wild-type; WRN, Werner; XPF, xeroderma pigmentosum complementation group F gene. ∗ Corresponding author. Tel.: +49 931 888 4089; fax: +49 931 888 4069. E-mail address: [email protected] (D. Schindler). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.05.006

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Genotype–phenotype correlations according to position and type of mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Identical mutations but divergent phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Mutations associated with prenatal and early postnatal lethality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Genotypes and other determinants compatible with long-term survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Somatic reversions and “mild” mutations correlate with exceptional longevity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Genetic alterations specifically associated with neoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Fanconi anemia as a segmental progeroid entity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Cellular phenotype as driving force of the FA progeroid features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Speculation: FA—a model of premature onset and accelerated progression of ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction There have been very few systematic studies dealing with genotype–phenotype correlations in FA, all of which have been retrospective (e.g. [1–6]). Following the discovery of genetic heterogeneity and cloning of the underlying genes, it was the (perhaps naïve) speculation that each of the FA complementation groups would present with more or less distinctive features. With a few notable exceptions (complementation groups FA-D1 and FAN), the initial expectation has not come to fruition: conspicuous genotype–phenotype correlations turned out to be the exception rather than the rule. In terms of effects on the clinical phenotype, it became clear that the type of the underlying mutation (e.g. frameshift vs. missense) is more important than the gene affected. While it has become straightforward to collect, sort and categorize molecular data, including functional testing of specific genetic alterations at the cellular and biochemical levels, the complexity of phenotypic features and their dependence on ethnic, genetic, ontogenetic, epigenetic, environmental and temporal interactions poses a much greater challenge. For example, homozygosity for the c.456 + 4A > T (IVS4 + 4A > T) mutation [7] in FANCC is associated with different degrees of phenotypic severity in the Ashkenazi Jewish compared to the Japanese population [8,9]. Likewise, early onset of bone marrow failure (BMF) cosegregates with the presence of severe malformations and vice versa, and with the effects of modifier genes [5,6,10]. A number of scoring systems has been developed to describe and classify the FA “phenome” in order to provide (in some sense binary) phenotypic information that is amenable to statistical treatments [1,2,5,6,11]. A very promising development is a recent study which explores and implements an FA phenotype scoring system based on a multivariate, non-parametric statistical method [12]. As a first result of this approach, Morales et al. [12] were able to show that mutations affecting different domains of the FANCA protein relate to different degrees of phenotypic severity. In terms of genotype–phenotype correlations in FA, we are a long way from a situation like that of the fibrillin, collagen or lamin A/C genes, where mutation type and position predict severity, organ system involvement and clinical outcome [13–15]. While aware of the incomplete status of genotype–phenotype correlations in FA, we will nonetheless attempt to offer some insights that we hope may guide additional research. 2. Multiple genotypes and multiple clinical phenotypes connected by a singular cellular phenotype On 15 December 2008, the Rockefeller Fanconi Anemia Mutation Database listed 622 unique and 1968 total patient mutations, unevenly distributed among 12 FA genes (excluding M,1 Fig. 1).

1 The status of FANCM as an FA gene and FA-M as a separate FA complementation group is currently a matter of debate.

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FA-A is the largest complementation group and includes the greatest number of distinct mutations due to a large number of private pathogenic sequence variants in FANCA. As shown in Fig. 1, patients of complementation groups FA-C and FA-G are the next most common, but the number of unique mutations in FANCC and FANCG is fewer. In contrast, FA-D2 and FA-D1 patients are rarer, but the number of unique mutations in these complementation groups is relatively higher. As illustrated in Fig. 2 (see Footnote 1), the high degree of genetic and mutational heterogeneity of FA corresponds to a remarkable variability at the clinical level: there are patients with a multitude of congenital malformations, patients with early childhood leukemia, medulloblastoma or Wilms tumor, and there are patients who reach adulthood with clinical and phenotypic features that are reminiscent of premature ageing. There are also adult patients with only subtle or no malformations, diagnosed as FA only because of infertility and short stature, or because they develop acute myelogenous leukemia (AML) or squamous cell carcinomas (SCCs). Some patients are diagnosed only because they show an abnormally severe reaction to chemotherapy of adult-onset malignancy. Clear-cut genotype–phenotype correlations are apparent only in a minority of FA genes, whereas in the majority of patients, the clinical phenotype cannot be predicted from the genotype. The important point of Fig. 2 is that the high degrees of heterogeneity at the molecular and phenotypic levels are in marked contrast to the FA cellular phenotype, which is surprisingly uniform. This uniformity of commonly examined cellular features (see below) extends well beyond increased spontaneous chromosome breakage as originally observed in FA cells [16]. With very few exceptions, the cellular phenotype can be safely predicted from any of the genetic alterations in any of the 12 known FA genes (see Footnote 1). FA cells are uniformly sensitive towards DNA-interstrand crosslinking (ICL) agents such as diepoxybutane (DEB), mitomycin C (MMC), cisplatin (CDDP) or nitrogen mustard (NM). This sensitivity is dose-dependent and manifests itself as reduced survival, elevated chromosome breakage rates, and cell cycle arrest. Ex vivo, FA primary lymphocytes [17], lymphoblasts [18], fibroblasts [19], erythroid progenitors [20], and primary bone marrow cells [21] of all complementation groups examined are uniformly sensitive towards atmospheric oxygen. At least under hypoxic culture conditions, FA fibroblasts of all complementation groups tested show a normal response to ionizing radiation [22]. With the exception of the FA-D1 and FA-N subtypes, FA cells are uniformly proficient in ICL-induced RAD51 foci formation. Consistent with the recessive mode of inheritance, the remarkable uniformity of the FA cellular phenotype requires the presence of bi-allelic (or, in the case of the X-chromosomal FANCB gene, hemizygous) mutations. Somatic reversion of constitutional mutations (revertant mosaicism) eliminates the features of the FA cellular phenotype in the reverted cell population. Because of extensive heterogeneity at the genetic and clinical levels, the classification of patients as FA sometimes solely depends on the presence of the characteristic cellular phenotype. Patients

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Fig. 1. Frequency distributions of unique (A) vs. total (B) FA gene mutations (see Footnote 1). Pie charts representing percentages are derived from the listing of 622 unique mutations devoid of patient information, and of 1968 mutations including multiple patient reports in the Rockefeller Fanconi Anemia Mutation database, now operated as locus-specific databases using the Leiden Open Source Variation Database software (LOVD v.2.0) and hosted by the Leiden University Medical Center, Leiden, The Netherlands (http://www.rockefeller.edu/fanconi/mutate/). For FA-A as the largest subtype group, percentages of both patients and FANCA mutations amount to 57% with a ratio of 1, indicating equal representation of private and recurrent mutations. For the next most frequent complementation groups (FA-C and FA-G), the ratio of unique vs. total mutations is <1 due to the relative prevalence of recurrent mutations in FANCC and FANCG. In contrast, many of the infrequent FA subtypes reveal ratios of close to 2:1 for unique vs. total mutations suggesting many different yet rare pathogenic sequence variants.

with other disorders may also be ICL-sensitive, but they display different sensitivity spectra (e.g. including sensitivity to ultra-violet light or ionizing radiation). Recent examples of partial overlap of the cellular phenotype but clinical phenotypes distinctive from FA include a patient with a unique combination of progeroid symptoms and photosensitivity due to a severe homozygous XPF mutation, or a patient with the cerebro-oculo-facio-skeletal (COFS) syndrome due to compound-heterozygous mutations in ERCC1, predicted to abrogate the XPF-binding domain of the protein [23,24]. If cells of any patient compatible with FA by clinical manifestations exhibit a typical “FA cellular phenotype”, the patient by definition is affected by FA, even if mutation analysis should fail to reveal a molecular defect in any of the known FA genes. This concept has also been the basis for the demonstration of genetic heterogeneity in FA and for the delineation of many FA complementation groups. In addition to showing causal mutations, there is no better definition of FA than by the cellular phenotype. Admittedly, we may not recognize FA forward mutations in single cell types, and we currently are unable to explain the basis of FA-like phenocopies, such as most cases designated as VATER or VACTERL association. Nonetheless, because of the uniqueness and diagnostic importance of the FA cellular phenotype, any discussion of genotype–phenotype correlations in FA must begin with a brief discussion of the various aspects of the cellular phenotype. 2.1. FA cellular phenotype: chromosome breakage and chromosome aberrations First reported in 1964 and 1965, increased spontaneous chromosomal breakage was recognized as a universal cellular attribute of FA [16,25]. Joenje et al. [17] have shown that the frequency of chromosome lesions in FA lymphocytes depends on the oxygen tension of the cell culture environment. There is every reason to believe that the spontaneous chromosomal instability of FA cells reflects their innate and unique sensitivity towards ambient (20%, v/v) oxygen concentrations that are standard in cell culture settings, whereas physiological oxygen tension for most of the tissues in vivo would correspond to oxygen concentrations of 3–5%. Chromosomal instability in FA involves mostly chromatid-type lesions, including chromatid interchanges (multiradials) that originate during S phase of the cell cycle. Chromatid breaks are microscopically visible consequences of unrepaired DNA double-strand breaks, occurring as

intermediates of ICL removal, while multiradial figures reflect successful but inaccurate repair of such lesions. In contrast to Bloom syndrome which is connected to FA at the molecular level [26], the breakpoints of interchanges are randomly distributed in FA involving mostly non-homologous chromosomes, with the notable exception of the sex chromosomes [27]. Since chromatid interchanges are the result of homology-directed DNA repair (HDR), the preferential involvement of non-homologous chromosomes in FA radial figures implies the genome-wide distribution of homologous DNA sequences serving as matrices for misrepair by error-prone HDR pathways [28,29]. While strongly elevated rates of sister chromatid exchanges (SCEs) [30] are the cytogenetic hallmark of Bloom syndrome cells, SCE rates are normal in FA but elevated in DT40 cells with recombinational deletions of FA genes (see also “The Fanconi Anemia Pathway: Insights from Somatic Cell Genetics Using DT40 Cell Line” Takata et al., this issue). Since this cell line of avian origin also lacks the p53 protein that cooperates with FA proteins in the maintenance of genomic integrity in human cells [31,32], caution is warranted when extrapolating findings obtained in the chicken model to the human situation. In addition to chromosome breakage, another chromosomal property that has repeatedly been evaluated in FA is telomere shortening [33–36]. Compared to controls, accelerated shortening of telomere repeats or enhanced loss of telomere signals of FA lymphocytes and serially propagated FA fibroblasts were frequently reported. Consistent with accelerated telomere shortening, Callen et al. [37,38] reported a >10-fold increase in chromosome end fusions in FA compared to normal control cells. It is difficult, however, to establish the biological significance of these findings, since attrition of telomere repeats is a physiological process during replication of cells that lack telomerase activity (see also “Fanconi Anemia Proteins and Endogenous Stresses” Pang and Andreassen, this issue). A comprehensive study of telomere dynamics revealed that both hematopoietic and nonhematopoietic FancG−/− mouse cells had normal telomere length, normal telomerase activity, and normal chromosome end-capping, even in the presence of extensive clastogen-induced genomic instability [39]. Murine telomerase-negative embryonic fibroblasts with human-like telomere length maintained normal telomeres when FancG was knocked down. Likewise, human primary FA-G fibroblasts showed no evidence for telomere dysfunction as assessed by different techniques. Cell loss and replication pressure may explain

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Fig. 2. The singular cellular FA phenotype. Multiple genotypes (see Footnote 1), as distinguished by specific genes and their range of mutations, and a broad variety of clinical manifestations, are unified pathogenetically by a conspicuous combination of cellular characteristics. ‘Unknown’ stands for FA patients whose gene defects cannot be identified at present. Some of the phenotype groups, if connected with particular mutations, are specified in Table 1. Color coding is for the purpose of illustration only and does not suggest functional relationships.

advanced telomere shortening of single FA cell populations [36]. Thus, different replicative histories of various cell types could be the reason for the divergent findings on telomere lengths in FA, secondary to the natural history of disease. At best, these findings reflect the accelerated “ageing” cellular phenotype of FA which, at least in peripheral blood cells, might be related to the precocious exhaustion of progenitor cell pools as the basis for BMF and the development of pancytopenia. Another prominent and clinically important feature of the FA cellular phenotype is the lineage-specific emergence of clonal chro-

mosome aberrations in bone marrow cells (see “Fanconi Anemia and its Diagnosis” Auerbach, this issue). The most frequent types of aberrations involve loss of the long arm or of the entire chromosome 7 (monosomy 7), gains of the long arm of chromosome 1, and gains of the long arm of chromosome 3 (reviewed by Neitzel et al. [40]). While some of the clonal chromosome changes may persist in FA patients for many years without conversion to myelodysplastic syndrome (MDS) or AML, monosomy 7 or gains of 3q26–3q29 indicate a high risk for the development of MDS and AML [41]. 3q26–3q29 gains or other structural aberrations including 3q26 are rather spe-

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cific for FA-associated or chemotherapy-induced AML [42]. Such aberrations result in amplification or transcriptional activation, and subsequent over-expression, of the ecotropic virus integration site 1 gene (EVI1). This gene encodes a zinc finger transcription factor with important roles in development and in leukogenesis [43]. EVI1 overexpression by itself has proven insufficient to cause leukemia, but it may maintain aberrant clones whose transformation is triggered by interaction with other factors, resulting in particularly aggressive forms of AML. 2.2. FA cellular phenotype: crosslinker sensitivity Less than 10 years after the discovery of spontaneous chromosome breakage in FA cells, Sasaki and Tonomura reported a high susceptibility of FA cells to chromosome breakage by ICL agents [44]. This seminal observation provided the basis for the “chromosome breakage test” as the gold standard for the pre- and postnatal laboratory confirmation of FA [45] (see “Fanconi Anemia and its Diagnosis” Auerbach, this issue). Despite initial concerns and reports of different degrees of sensitivity towards the various ICL agents [46], there is now agreement in the field that any deviations in the chromosome breakage rates between different ICL agents reflect dissimilarities in drug activity, stability and metabolism, and in laboratory procedures, rather than intrinsic biological differences. Regardless of whether a given laboratory employs DEB, MMC, NM, CDDP, melphalan, psoralen-UVA or others, and regardless of whether lymphocytes, bone marrow cells, fibroblasts or amniotic fluid cells are tested, if properly executed and controlled, the test result is likely to differentiate between sensitive and insensitive cells. This is best exemplified by the phenomenon of revertant mosaicism, where ICL-sensitive and ICL-insensitive cell populations may reside side by side within the same cell culture specimen [47]. In addition to the chromosome breakage test, cell cycle and cell survival assays are routinely used for the qualitative and quantitative determination of ICL sensitivity. Increased accumulation in G2 after exposure to an ICL agent is in the same way specific for FA cells as are enhanced chromosome breakage rates. Comparative studies have provided matched results [48,49]. All three cellular endpoints, chromosome breakage, G2 accumulation and survival can be considered diagnostic. Semi-automated techniques such as cell cycle recording and analysis facilitate studies that include complementation trials of cells from FA patients for several FA genes in parallel (e.g. [50,51]). An example of MMC sensitivity testing using the cell cycle assay is shown in Fig. 3 where cultures containing a mixture of native FA and isogenic but ex vivo complemented fibroblasts of subtype G were exposed to increasing concentrations of MMC. Regarding G2 phase accumulations (as a convenient measure of MMC sensitivity), the mutant cells clearly are more sensitive than their complemented counterparts within a sufficiently wide diagnostic range. In terms of genotype–phenotype correlations, the assessment of ICL sensitivity provides the basis for the interpretation of functional consequences of a given genetic alteration. An exemplary study in that regard is the work by Adachi et al. [52] who tested the MMC sensitivity of 21 patient-derived FANCA missense mutations or small in-frame deletions. While mutants lacking protein expression (null mutants) uniformly show MMC sensitivity, the situation is less clear for single amino acid substitutions which may retain variant degrees of altered protein function or even represent functionally silent changes. As a case in point, five of the alterations tested by Adachi et al. [52] were MMC-resistant, four were mildly, and twelve were highly sensitive. Reconstitution of the FA pathway – in terms of phosphorylation and nuclear localization of FANCA, interaction with other proteins of the FA core complex (FA complex I [53]), and monoubiquitination of FANCD2 – correlated inversely with the degree of MMC sensitivity. The authors speculated that variant degrees of cellular MMC sensitivity might reflect

Fig. 3. MMC-induced G2 phase arrest of FA fibroblasts. Subcultures of fibroblasts derived from an FA-G patient were transduced with a retroviral vector containing wtFANCG cDNA in a bi-cistronic construct with enhanced green-fluorescent protein (GFP) cDNA. These cultures were exposed to different concentrations of MMC for 48 h. Cells without (left) and with (right) successful gene transfer were gated by flow cytometry according to the absence (left) or presence (right) of green fluorescence. At each MMC concentration, Hoechst 33342-stained cells that have retained the FA genotype (left) display much higher G2 phase peaks than cells whose genotypes were corrected by transduction with wtFANCG cDNA (right). Reproduced from [83].

the functional diversity of the different kinds of genetic alterations and thus may account for the variability of clinical phenotypes [52]. Unfortunately, such allele testing is reliable only with isogenic or otherwise artificial read-out systems. In other settings, there is no consistent correlation between levels of chromosome breakage and severity of the clinical phenotype [49]. Another limitation is that Adachi et al. [52] expressed their various mutants in SV40-transformed fibroblasts. Since SV40 transformation is known to affect some of the physiological functions of cultivated cells [54], the observation of variant degrees of MMC sensitivity in such transformed cells must be viewed with caution. Allele testing in immortalized lymphoblastoid cell lines (LCLs) appears to be more reliable and is a convenient procedure, especially when combined with retroviral vector techniques. An example is illustrated in Fig. 4. The amino acid substitution p.P184Q [7] was initially described by Ameziane et al. [55] as a (presumably pathogenic) missense change in the FANCE gene in a patient whose second mutation remained elusive. As the pathogenicity of the p.P184Q variant was controversial, we transduced an FA-E null LCL with a retroviral construct containing site-mutated FANCE cDNA in order to express the substitution p.P184Q [Neveling et al., in preparation]. MMC sensitivity was restored to a level similar to that of cells transduced with wt FANCE cDNA, indicating the innocuous nature of this particular variant. As a negative control, the expression of enhanced green-fluorescent protein (GFP) in a corresponding construct failed to restore MMC sensitivity in the same FA-E null cell line. Failure to complement MMC sensitivity would also indicate the dysfunctional nature of any questionable genetic alteration. In addition to allele testing, retroviral transfer of wt FA cDNAs into different types of cultured cells is now widely used as an expedient approach to subtyping of FA patients by the determination of cellular ICL sensi-

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vation of FA genes sensitizes tumor cells to ICL agents, suggesting a promising approach to tumor therapy [72–75]. Of practical importance to FA patients is the suggestion of endogenous formation of ICLs by products of lipid peroxidation [76,77] and as byproducts of normal digestion [78]. 2.3. FA cellular phenotype: cell cycle disturbance

Fig. 4. Testing of variant alleles. Cell cycle distributions of a FANCE null lymphoblast cell line. Subcultures were transduced with different constructs in S11-type oncoretroviral vectors [56] and express either enhanced green-fluorescent protein (GFP) alone, in combination with wtFANCE, or FANCE with the missense substitution FANCE P184Q. Cells that express GFP alone retain their FA-characteristic G2 phase accumulation after exposure to MMC (49.5%). G2 phase peak size following exposure to MMC is reduced after transduction with wtFANCE cDNA (9.3%), similar to that of untreated cells. Site-mutated (p.P184Q [7]) FANCE complements the cell cycle defect to nearly the same degree (G2 phase, 9.9%), indicating the harmless nature of this particular amino acid substitution (Neveling et al., in preparation).

tivity via chromosome breakage, cell cycle, or cell survival analysis (e.g. [50,51,56]), including the characterization of candidate genes as novel FA genes (e.g. [57]). ICL sensitivity is universal, but not exclusive to FA cells. There are a number of reports describing clinical and cellular phenotypes that are suggestive of, but not fully compatible with FA (e.g. [24,58–60]). One such report [60] describes the highly unusual situation of a patient with an unclear disease entity whose lymphocytes showed some degree of spontaneous chromosome breakage and MMC sensitivity whereas the findings in his fibroblasts were borderline at best. While it was not unexpected that Seckel syndrome cells with ATR mutations would display MMC sensitivity [61], ICL sensitivity came as a complete surprise in cells of patients with Cornelia-de-Lange syndrome for which there was no previous indication of impaired HDR [62]. A significant overlap with regard to cellular and clinical phenotypes has been repeatedly noted among patients affected by FA and patients suffering from the Nijmegen breakage syndrome (NBS) [58,59,63]. In addition to features such as short stature and microcephaly, the overlap includes MMC sensitivity. Despite this partially shared cellular phenotype, FA and NBS can be clearly differentiated on the basis of cellular sensitivity towards ionizing radiation, which is pronounced in NBS. In contrast, FA fibroblasts of different subtypes were shown to be normally sensitive to ionizing irradiation in terms of clonogenic survival [22]. Other assays are suggestive of some minor degrees of radiosensitivity under ambient oxygen conditions [64,65]. Any cellular phenotype that includes ICL sensitivity rather than radio-sensitivity represents a potential candidate for a defect of an as yet unknown FA gene and should be tested for its proficiency of the FA/BRCA pathway (e.g. [66–69]). Knock-down of a number of different genes results in cellular MMC sensitivity. Some of the corresponding proteins are potential or proven members of the FA core complex (such as FAAP100), or otherwise involved in the FA/BRCA pathway [70]. Lastly, a number of different tumor types arising in non-FA patients exhibit ICL sensitivity, and a defective FA/BRCA pathway could be demonstrated in some of these (reviewed by [71]). In fact, inacti-

Many of the phenotypic features of FA, including pre- and postnatal growth retardation, organ hypoplasia, BMF, and features of premature ageing may, at least in part, be explained by impaired cell proliferation as a fundamental, albeit variably expressed defect. Consistent with this notion, an alteration of cell cycle transit was demonstrated by Dutrillaux et al. [79], who observed a prolongation of G2 phase duration in short-term cultures of FA peripheral blood lymphocytes. This key observation was confirmed and extended by Kubbies et al. [80] using high resolution BrdU-Hoechst versus ethidium bromide flow cytometry. This unique technique permits the quantitative assessment of compartment-specific cell cycle progression of mitogen-responsive cells throughout four consecutive cell cycles [81]. By fitting the resulting data sets to a modified transition probability model of cell kinetics [82], compartment-specific cell cycle delay and compartment-specific cell cycle arrest can be derived with unprecedented precision. As shown in Fig. 5, lectinstimulated (but otherwise untreated) FA lymphocytes display a distinctive cell kinetic phenotype with prominent accumulations of cells within the G2 phase compartments of consecutive cell cycles (shaded areas in Fig. 5). These spontaneous accumulations consist of a mixture of cells whose G2 phase transit is either delayed or completely arrested. Comparatively minor delay and arrest of a small fraction of FA cells within the S phase compartment of the cell cycle are revealed by careful quantitative analysis [80,83]. Taken together, these high resolution cell kinetic data provide proof that the cell cycle disturbance of untreated FA cells grown under ambient oxygen conditions predominantly affects the G2 and, to a much lesser degree, the S phase compartments of the cell cycle, with the majority of cells being delayed or permanently arrested in G2. This phenomenon is enhanced in situations where FA cells are challenged by MMC and excessive unrepaired DNA damage accumulates. Since the cell cycle disturbance in FA is related to the underlying genetic defect and principally affects all cell types, hematopoietic stem cells or progenitor cell lineages may sustain severe deficits of their cycling cell fractions. The attendant risk is premature exhaustion of the respective progenitor and stem cell pools, as seen in the aplastic phase of FA. While it is very clear that impaired FA gene function leads to the accumulation of DNA damage throughout the S and G2 phases of the cell cycle, what has been less clear in the literature is where precisely and by which mechanism these defective cells are arrested. In fact, most papers talk of “G2/M” arrest as a typical feature of the FA cell cycle. In order to distinguish between G2 and M arrest, we used phospho-histone H3 (H3P) staining that specifically identifies cells that have completed G2 and have entered mitosis [83]. Fig. 6 illustrates that exposure of FA cells to MMC strongly increases the H3P-negative portion of the 4n (G2/M) compartment but decreases the H3P-positive 4n cell fraction. In contrast, exposure to a known spindle-disrupting agent (nocodazole) elevates the H3P-positive 4n (G2/M) cell fraction and, by no more than that proportion, the total 4n compartment. These experiments prove that the FAcharacteristic cell accumulation occurs prior to mitosis. Therefore, the FA cell cycle defect is correctly designated as G2 phase arrest. Akkari et al. [84] have argued that because exposure of FA cells to ICL agents after completion of S phase produced neither G2 phase arrest nor chromosome breakage, ICL-containing cells must be arrested in late S, prior to G2 transition. However, flow-cytometric studies uniformly yield a 4n DNA content of the arrested cell population

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Fig. 5. Kinetic display of successive cell cycle compartment transitions. Shown are curve fits of semi-logarithmically plotted cell cycle distributions from PHA-stimulated lymphocyte cultures. Plotted are the proportions of cells remaining in any given compartment as a function of time after culture initiation (␣-plots according to Smith and Martin, modified as proposed [82]). The left hand graph reflects a culture derived from an FA patient, the right hand graph that of a non-FA control. Distances between intercepts of the exit curves with the x-axis (representing minimum S, G2 and G2 durations) are indicated by headline bars. The corresponding arrest fractions are given as vertical bars between the intercepts of the same curves with the y-axis. Minimal compartment transit times of the first cycle are 10.4 h (S) and 5.6 h (G2) in the FA vs. 9.4 h (S) and 3.4 h (G2) in the control culture. Arrest fractions amount to 4.2% (S) and 16.2% (G2) in the FA vs. 1.2% (S) and 1.5% (G2) in the control culture. For the purpose of illustration, the G2 phase compartments are shaded. Modified from [83].

[48,49,85,86], indicating completion of DNA replication. On the basis of DNA content distributions, there is no evidence for massive accumulations of FA cells during S phase. Moreover, systematic studies with the induction of ICLs via psoralen/UVA treatments have shown that FA cells fail to arrest in S phase, and DNA synthesis continues in these cells despite the presence of ICLs [87]. Current concepts suggest that the FA/BRCA pathway senses nonrepaired ICLs through stalled replication forks, occurring in S phase [53]. This notion may explain the finding that FA cells exposed to ICL agents after S phase do not show G2 arrest [84]. Spontaneous DNA damage in FA cells, and damage induced by ICL agents appear not merely of the type that would activate the intra-S checkpoint. Whether the S phase checkpoint is inefficient or partially defec-

tive in FA cells remains an unresolved question. Some observations [87–89] report an S phase checkpoint defect restricted to a subset of complementation groups [88] or to a single time point after ICL induction [87], but cannot reconcile these short-term findings with long-term reduction of DNA synthesis in FA compared to control cells [89]. At any event, all evidence indicates that FA cells with ICLs activate the G2/M checkpoint such that they are halted in G2 to prevent entry of damaged cells into M phase, thus allowing for post-replication repair. In terms of the underlying mechanism, the observation that G2 arrest depends on the function of both ATR and CHK1 emphasizes primary usage of the G2/M checkpoint signal transduction pathway by FA cells [90]. A preserved function of the G2/M checkpoint in FA cells has been convincingly demonstrated by comparative cell cycle studies using ICL agents, hydroxyurea, and ionizing radiation. These studies have shown that the accumulation of FA cells in G2 reflects an essentially normal cellular response to excessive and persisting DNA damage [49,91,92]. Exposure of FA cells to the G2/M-checkpoint inhibitor caffeine all but eliminates the FA-typical G2 phase prolongation and arrest. Caffeine-treated FA cells progress through G2 and mitosis without any delay, at the cost of highly elevated rates of chromosome aberrations, and decay during the following G1 phase [93–95]. These types of experiments provide additional evidence for a well-conserved G2/M checkpoint function in FA cells. For its activation, ICL agents forming covalent bonds with the DNA duplex strands are highly specific. Compounds that mimic ICLs by a covalent bond to one strand and hydrogen bonding to the opposite strand, such as trabectedin, fail to induce G2 arrest [96]. 2.4. FA cellular phenotype: oxygen sensitivity

Fig. 6. Flow-cytometric differentiation between G2 and M phase arrest. FA-D1 lymphoblasts respond to exposure to 45 nM MMC for 48 h with a disproportionate increase of their 4n (G2/M phase) cell cycle fraction as shown by DNA histograms recorded after Hoechst 33342 staining (upper panel, 14.6 → 37.8%). Concomitantly, H3P-positive (mitotic) cells are reduced to less than half (superimposed scatter plots above the G2 phase peaks, 1.4 → 0.6%). The inverse relationship between MMCinduced 4n accumulation and H3P expression indicates that the FA-typical G2 arrest is pre-mitotic. In contrast, exposure of normal control lypmphoblasts to 1.5 ␮M nocodazole for 24 h markedly increases the H3P-expressing (mitotic) cell fraction (lower panel, 0.7 → 16.5%). This is accompanied by a proportional expansion of the total 4n (G2/M) compartment (7.6 → 15.3%). In FA cells the effect is similar (not shown), indicating that nocodazole, but not FA plus MMC, precipitates mitotic arrest.

Hypersensitivity of FA cells to ambient oxygen culture conditions was first demonstrated by Joenje et al. [17], who discovered that spontaneous chromosome breakage rates of FA peripheral blood T lymphocytes varied as a function of oxygen tension of the cell culture environment. These studies were confirmed and extended to primary FA fibroblasts [19,97]. It was shown that poor ex vivo growth and cloning efficiency of FA fibroblasts could be restored to near normal if these cultures were established, maintained and serially propagated at hypoxic (5%, v/v) rather than ambient (20%, v/v) oxygen concentrations [19]. It was also shown that these improvements were, in part, due to reductions of the spontaneous G2 accumulations which characterize FA cells grown under ambient oxygen conditions [98,99]. As an additional cell type, LCLs derived from FA patients were shown to be oxygen-sensitive

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[18]. Since the degree of oxygen sensitivity of this cell type varied as a function of the iron concentration in the culture media, FA cells obviously are more sensitive to reactive oxygen species (ROS) produced by Fenton-type reactions [18]. With regard to native FA bone marrow specimens, Alter et al. [20] and Cohen-Haguenauer et al. [21] were able to show that exposure to low oxygen improved the colony-forming ability of hematopoietic progenitor cells. Indirect evidence for the mutagenic activity of ambient oxygen was obtained by passing reporter plasmids (“shuttle vectors”) through FA cells, grown at different oxygen concentrations [100]. Collectively, these early descriptive data appear highly relevant for the explanation of both genetic instability and BMF in FA, since hematopoietic stem cells apparently require protection by a low-oxygen niche [101], and excessive ROS levels within these highly susceptible cells may cause genetic instability, loss of self-renewal, and apoptotic cell death [102,103]. Pagano et al. [104] have made an attractive, albeit (in terms of experimental evidence) still unsubstantiated case that many of the clinical and cellular abnormalities observed in FA, including congenital malformations, BMF, pigmentation changes, insulin resistance and propensity to neoplasia may result from ROS-induced damage which, in a situation of defective caretaker genes, cannot be handled properly. A number of recent studies by the Qishen Pang laboratory (see “Fanconi Anemia Proteins and Endogenous Stresses” Pang and Andreassen, this issue) and others provide evidence for the likely involvement of FA genes in oxidative metabolism and oxygen-dependent regulation of cell functions, including mitochondrial function [103,105–107]. A practical consequence of the oxygen-sensitive FA cellular phenotype is that drug sensitivity or DNA damage and repair assays, such as the comet assay, may yield biologically divergent results if they are performed under different oxygen conditions [64,108]. 2.5. FA cellular phenotype: deregulated apoptosis A pro-apoptotic phenotype of proliferating cells has been associated with the pathogenesis of BMF and with the emergence of malformations during embryonic development in FA. Increasing evidence suggests that enhanced oxidant- and myelosuppressive cytokine-mediated apoptosis of hematopoietic stem and progenitor cells contributes to the depletion of these cell types. Bijangi-Vishehsaraei et al. [109] studied increased apoptotic signalling through the redox-dependent protein, apoptosis signal-regulating kinase 1 (Ask1). They demonstrated that tumor necrosis factor (TNF)-␣ induces hyperactivation of Ask1 and of the downstream effector p38 in FancC−/− murine embryonic fibroblasts (MEFs). Ask1 inactivation in FancC−/− MEFs or hematopoietic progenitor cells restored survival to wt levels despite the presence of TNF-␣. In addition, targeting the Ask1 pathway by antioxidants or a p38 inhibitor protected FancC−/− MEFs and c-kit+ cells from TNF-␣-induced apoptosis. These experimental data suggest that the predisposition of FancC−/− hematopoietic progenitors to apoptosis is mediated, at least in part, through altered redox regulation and Ask1 hyperactivation. Li et al. [110] showed a time-dependent increase in apoptosis of primitive progenitors in ex vivo bone marrow cultures of FancC−/− mice under conditions that promote the proliferation of wt stem/progenitor cells. The intrinsic defect of FancC−/− stem/progenitor cells provided selective pressure such that a subset of cells which had lost sensitivity to apoptosis gained propensity for the evolution of clonal hematopoiesis. BMF in FA patients related to excessive apoptosis of hematopoietic progenitor cells has also been associated with an over-production of inhibitory cytokines such as TNF-␣ and interferon-␥ in vivo or ex vivo [111]. Over-production of myelosuppressive cytokines and hypersensitivity of FA cells towards their effects enhance the production of ROS and up-regulate the Fas death receptor [112–114]. Implication of the Fas pathway in the disruption of

apoptotic control has been suggested [115]. Of note, death of FA cells in response to ICL agents has been attributed to a mechanism involving necrosis rather than apoptosis [116]. Increased spontaneous apoptosis in FA exerts its deleterious action during ontogenesis. In zebrafish (see “The Fanconi Anemia/BRCA Gene Network in Zebrafish: Embryonic Expression and Comparative Genomics” Titus et al., this issue), Fancd2 was shown to be essential during embryogenesis to prevent inappropriate apoptosis in neural cells and other tissues undergoing high rates of proliferative expansion [117]. Fancd2-deficient zebrafish develop an FA-like pattern of malformations due to extensive spontaneous apoptosis. Increased apoptosis and developmental defects were reversed by injection of human FANCD2 or by knock-down of p53, indicating that spontaneous apoptosis in FA cells is p53-dependent. Using murine models of FA (see also “Mouse Models of Fanconi Anemia” Parmar et al., this issue), Sii-Felice et al. [118] studied microcephaly in FancA−/− and FancG−/− mice. They examined neural stem and progenitor cells during developmental and adult neurogenesis. Absence of FancA or FancG provoked neural progenitor apoptosis during forebrain development, and this persisted during adulthood, leading to depletion of the neural stem cell pool with ageing. Post-mitotic neurons in the neocortex were not involved. 2.6. FA cellular phenotype: RAD51 foci formation Nuclear (repair) foci following exposure to genotoxic agents are thought to represent macromolecular assemblies of various proteins recruited to sites of DNA damage [119,120]. Since RAD51 is the key recombinase in HDR, RAD51 foci formation was repeatedly tested in FA cells of various complementation groups. Initial studies claimed overall reduced RAD51 foci formation in FA cells [121], but it was later shown that cells from most FA complementation groups have normal rates of DNA damage-induced RAD51 foci formation [122]. The only exceptions so far are the FA-D1 and FA-N subtypes where <10% of cells are RAD51 foci-positive [57,122]. This exceptional RAD51 foci phenotype confirms the close physical and functional relationship between the BRCA2 and PALB2 proteins that are instrumental in recruiting the RAD51 recombinase to sites of HDR. A case in point is the recent observation that core- and ID complex-defective (complex I and II, respectively [53]) FA cells are impaired in the initial excision steps of ICL removal, in contrast to FA-D1 cells whose defect appears to reside in the later recombination step [123]. These findings confirm the distinctive nature of the FA-D1 and FA-N cellular phenotype as suggested by rates of spontaneous chromosome breaks and sensitivity to ICL agents, which are above average compared to FA cells of the other complementation groups. This notable exception from the general rule of a “singular” cellular phenotype in FA fits well with the early tumor proneness that is hallmark of the clinical phenotype of D1 and N patients [124]. 3. Genotype–phenotype correlations according to complementation group 3.1. Distinctive clinical phenotypes as function of complementation group If one attempts a (rather arbitrary) grouping of major FA phenotypes according to their clinical features (Table 1), it is evident that all of these groups are represented by more than a single FA gene. It is also evident that some of these phenotype groups are represented by a limited number and by different members of the FA genes. For example, the phenotypic category with the least severe clinical outcome in this tabulation is devoid of patients belonging to complementation groups B, E, and F. A similar selective situation exists for the “hematological improvement phenotype” that is caused by somatic reversions. So far, revertant mosaicism has been

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Table 1 Distinctive phenotype groups in FA and examples of corresponding mutations. Phenotypea

Key features

FA genec

Mutatione /predicted effect

Reference

Zero or low CABb score, survival to adulthood, stable blood counts, occasional fertility

A C D1 D2

c.3788 3790delTCT/p.1263delF c.67delG/p.D23IfsX23 c.8488-1G > A/p.W2830 K2833del c.2444G > A/p.R815Q

[192] [145] [139] [137]

Hematological improvement

Somatic reversions resulting in revertant mosaicism

A L D2 D2 D2 N

c.856C > T/p.Q256Xg back mutation c.483delATCAC/p.E161DfsX25g comp. mutation c.1948-16T > G/p.E650Xg comp. mutation c.2775 2776CC > TT/p.R926Xg recombination c.2775 2776CC > TT/p.R926Xg back mutation c.1802T > A/p.Y551Xg Alu mediat. recombination

[193] [125] [137] [137] [137] [194]

Low MCAb , high neoplasia

Low CABb score, late BMFb

A A D2

c.1549C > T/p.R517W c. 2807A > G/p.E936G c.458T > C/p.L153S

[149] [181] [154]

High MCAb , low neoplasia

High CABb score, early BMFb

D2 I J

Most of the mutations in FANCD2 and FANCI c.2533C > T/p.R798X

[137] [138] [195]

A (3%)d C (15%)d E (40%)d F (30%)d G (8%)d D1 (33%)d

n.a.f IVS4 + 4 > T (c.456 +4A > T)/p.G116 N152del n.a.f n.a.f n.a.f 1584del4, probably identical with c.1311 1314delAGAT/p.K437NfsX21

[196] [196] [196] [196] [196] [196] [55]

Close to normal

VA(C)TER(L)-like

Vertebral defects, anal/duodenal atresia, (cardiac defects), tracheo-esophageal fistula/atresia, renal defects, (limb defects)

VACTERL-H

Plus hydrocephalus

B

c. 1496+5G > A/p.K443VfsX4

[197]

Early solid tumor/leukemia

Medulloblastoma, Wilms tumor, AMLb

D1 N

IVS7+2T > G (c.631+2T > G)/p.G173SfsX19 c.3549C > A/p.Y1183X

[198] [57]

a b c d e f g

The phenotype categories are explained in Fig. 2. Abbreviations: AML, acute myelogenous leukemia; BMF, bone marrow failure; CAB, congenital abnormalities; MCA, multiple congenital anomalies. See Footnote 1. Prevalence in the corresponding complementation group. Nomenclature of mutations: Human genome variation society, http://www.hgvs.org/mutnomen/. n.a., not available. mechanism of revertant mosaicism.

documented only for 5 of the 12 currently proven FA genes (see Footnote 1) [125]. Since some of the FA subtypes are exceedingly rare and represented by very few or even single patients, the seemingly selective involvement of FA genes in these specific phenotypes might be due to ascertainment bias and, thus, spurious indeed. With regard to the cumulative incidence of the frequent FAassociated neoplasms (AML and SCC), a prognostically important distinction has been established. FA patients with few congenital anomalies and normal radial ray structures are at higher risk, in contrast to patients with radial ray defects and multiple congenital anomalies (MCA) who are at lower risk for those malignancies [5,6]. Whether this correlation applies to members of all complementation groups (with the obvious exception of FA-D1 and FA-N patients) remains to be substantiated, given the uneven representation of the various FA subtypes in these studies. Regarding patients with the low MCA and high risk of neoplasia phenotype, what we urgently need to know is whether susceptibility to the feared SCC, in addition to factors such as HSCT, is influenced or even determined by a specific genotype. Subtyping and mutation analysis is of utmost importance in these patients. FA-D1 and FA-N patients represent the only convincing examples of a genotype–phenotype relationship in which the affected gene appears to play a greater role than the underlying type of mutation. Unlike patients of any other subtype, FA-D1 and FA-N patients are characterized by very early onset of medulloblastoma, Wilms tumor or AML, with a cumulative incidence probability of malignancy amount to 97% by the age of 5.2 years [124]. Indeed, epidemiological data and work with mouse models suggest that normal FANCD1/BRCA2 function is required for the suppression of medulloblastoma and hematological malignancies [126,127].

3.2. With few exceptions, primacy of type of mutation over type of gene There are two large studies that tried to find associations between complementation group and clinical outcome. The European study [2] examined 245 patients from 7 complementation groups. It concluded that FA-G patients had more severe cytopenia and a higher incidence of leukemia compared to the other groups, and that somatic abnormalities were less prevalent in FAC patients compared to the rarer groups FA-D (meaning D2), FA-E and FA-F. Earlier BMF in the group of FA-G patients may in part be due to cases of neonatal pancytopenia in FA patients carrying the c.1649delC mutation [7] in FANCG [128]. The American study summarized data of 754 patients (not all of them subtyped) that were followed for up to 20 years by the International Fanconi Anemia Registry (IFAR; [3]). The IFAR study reported a significantly earlier onset of BMF and poorer overall survival for FA-C compared to FA-A and FA-G patients, the exact opposite of the result of the European study. This obvious discrepancy is readily explained by the fact that the IFAR cohort contains many more FA-C patients with the clinically severe intron 4 (c.456 + 4A > T) and exon 14 (c.1642C > T) mutations [7] in FANCC. In contrast, patients with the clinically less severe c.67delG mutation [7] prevail in the European cohort. There is no better way to demonstrate the futility of trying to correlate clinical phenotypes to individual FA genes rather than to individual mutations, including somatic reversion of constitutional mutations. A higher frequency of revertant mosaicism in subtype FA-A might indeed explain the earlier occurrence of BMF in subtypes FA-C and FA-G. After adjusting for radial ray malformations and congenital abnormality (CAB) scores which were previously

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shown to have a strong influence on tumor risk [5], the Rosenberg et al. study [6] reports higher rates of BMF for FA-C compared to FA-A patients (relative hazard = 5.4, 95% CI = 2.0–14.6, p = 0.001). The BMF rates for FA-G relative to FA-A patients were intermediate. Clearly, in order to interpret this type of study in terms of clinically useful genotype–phenotype correlations one would need to know the particular types of mutations and their potential for somatic reversion. As discussed below, the amount of information on the clinical outcome of individual mutations is still fragmentary but expected to improve during the coming years due to technical advances and reduced costs of DNA sequencing [55]. A major future achievement would be manifestation-related, e.g. tumorspecific, rather than merely locus-specific mutation databases in FA. 4. Genotype–phenotype correlations according to position and type of mutation 4.1. Identical mutations but divergent phenotypes There are a number of reports describing divergent phenotypes among siblings, naturally in the presence of identical mutations [129–132]. These examples illustrate how phenotypes are influenced by factors other than the underlying genetic alteration. A particularly instructive example is the case of two sisters, portrayed in Fig. 7. Despite identical FANCA mutations (a large and a small deletion: EX15 20del and c.3788 3790delTCT, resulting in p.F1263del [7]), both phenotypes and clinical courses of these siblings were markedly different, and this difference remains largely unexplained. Failure to recognize revertant mosaicism might account for such discrepancies in some of the early reports (e.g. [133,134]), a suggestion fostered by recent molecular studies [135]. Apart from the clinical experience that phenotypes of index cases are often more severe [136], differential genetic background, influences of modifier genes, environmental factors and chance effects have been invoked to explain such striking discrepancies (see also “Fanconi Anemia and its Diagnosis” Auerbach, this issue). More definite answers will have to await the results of prospective studies.

4.2. Mutations associated with prenatal and early postnatal lethality One of the most informative criteria in genotype–phenotype correlations is whether a given type of genetic alteration is compatible with intrauterine survival, and whether a patient survives to adulthood. Viewed collectively, patients belonging to subgroup FA-D2 are, on average, more severely affected and experience a more rapid hematological course regardless of whether or not some individuals survive to adulthood [137]. More serious manifestations prevail even though the combinations of mutations occurring in FA-D2 and probably in FA-I patients do not result in complete loss of protein function [138]. This suggests that bi-allelic loss-of-function mutations in FANCD2 and FANCI may be lethal in humans [137,138]. In support of this notion, variable levels of residual protein have been convincingly demonstrated in all tested FA-D2 cell lines. The emergence of the monoubiquitated FANCD2-L isoform, inducible in different cell types in a time- and dose-dependent manner, implies preservation of residual protein function [137]. A similar situation has been suggested for subgroup D1 due to mutations in BRCA2 [139]. However, in the absence of systematic fertility data and abortion histories of FA-D1 families, evidence remains indirect. The BRCA2 mutation frequency in the general breast cancer population in the US, unselected for family history, has been estimated to be 2.3% [140]. Assuming that 11% of all women develop breast cancer and that breast cancer-negative women do not contribute to the BRCA2 mutation frequency, the proportion of women heterozygous for BRCA2 mutations would amount to 2.5 per 1000. Given an equal frequency in men, this figure would be characteristic of the general population and would result in a predicted prevalence of FA-D1 patients of 1.5 per 1,000,000. This proportion would make up approximately 50% of all FA cases (about 3 per 1,000,000 [141]). Although FA-D1 (and FA-N) patients show the shortest average lifespan of all FA subgroups, the predicted frequency of FA-D1 patients appears at great variance with current estimates, which amount to only 2–3% [53] for subtype D1 among FA patients (cf. Fig. 1). This discrepancy suggests overwhelming prenatal mortality associated with the FA-D1 genotype. Since BRCA1 and BRCA2 are involved in related pathways and

Fig. 7. Discordant phenotypes and clinical courses of two FA siblings. The left photograph shows the elder sister at age 14 with palor from anemia and turricephaly due to long-term transfusion dependency. The right photograph, by contrast, shows the younger sister without visible signs of the disease despite her older age of 17 when the picture was taken, but with some manifestation of androgen therapy. Distinctive features are specified below the photographs. Notwithstanding their different histories and courses, both sisters succumbed to their disease at similar ages, the elder severely affected at age 21, the younger less affected at age 20 years. Courtesy of Ralf Dietrich and Cornelia Sowa-Dietrich.

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Fig. 8. Distributions of FANCD1/BRCA2 mutations within the gene. (A) Bi-variate positional plot of bi-allelic BRCA2 mutations in FA-D1 patients. Of the 27 patients in the review by Alter et al. [124], which includes all cases reported in the literature at that time, three cases were omitted, one that was not genotyped and two (HSC230 and 900/1) that had unrelated/incomplete disease genotypes: HSC230 has been re-classified to group FA-B, and in IFAR 900/1 the second authentic BRCA2 mutation remained elusive. In addition, the bi-variate plot (A) includes another more recent case from the literature [55] and four unpublished cases from our own observations. Closed circles represent patients without (n = 23), open circles those with one missense allele (n = 6). Gray circles apposing black ones represent siblings with identical mutations. FA-D1 patients with homozygous mutations (n = 6) are located along the dotted diagonal. All cases are labeled as designated in the references, the unpublished cases as FA1A/1B to FA3. The projected exon 11/exon 11 square highlights that no bi-allelic exon 11 BRCA2 mutations have been reported to date. (B) The complete listing of all breast cancer-associated heterozygous BRCA2 sequence variants in the Breast Cancer Information Core database (http://research.nhgri.nih.gov/bic/) at the end of 2008 contained 11,328 entries. These were assigned to BRCA2 exons 2 through 27 resulting in a univariate positional plot (histogram). Its columns represent mutations per nucleotides (height, y-axis) for the individual exons that, in turn, are graphically displayed by their range (BRCA2 position, x-axis).

heterozygous mutation carriers show similar frequencies and manifestations, this raises the question of whether individuals with bi-allelic BRCA1 mutations might exist. Assuming that such individuals were affected with a type of FA comparable to or indeed more severe than that of FA-D1 patients, they could be exceedingly rare or even subject to complete prenatal loss. Evidence for prenatal selection also derives from the distribution of BRCA2 mutations observed in FA-D1 patients. Fig. 8A illustrates the scattering of mutated alleles in a series of 29 FA-D1 patients: 24 cases are from the review of Alter et al. [124], one completely genotyped case is from Ameziane et al. [55] and 4 unpublished cases are from our own observations. As shown in Fig. 8A, 23 of the 58 mutations (39.7%) are located upstream of exon 11, 17 (29.3%) are within exon 11 (encoding the functionally crucial BRCT repeats) and 18 (31.0%) are downstream of exon 11. This distribution of BRCA2 mutations in FA-D1 patients strongly deviates from that of heterozygous mutation carriers affected by breast cancer, as provided in the Breast Cancer Information Core (BIC) database (http://research.nhgri.nih.gov/bic/) (Fig. 8B). At the end of 2008, the latter distribution revealed 18.1% (n = 2056) of mutations in exons 1 through 10, 46.7% (n = 5287) within exon 11 and 35.2% (n = 3985) in exons 12 through 27, which is significantly different from that in FAD1 patients (p < 0.001, chi square, 2 dof). Since close to 50% of BRCA2 mutations of heterozygous breast cancer patients are located in exon 11, one might expect that approximately 25% of FA-D1 patients should have bi-allelic mutations in exon 11. In fact, there are none, a situation first noticed by Popp et al. [142] and highlighted by the projected exon 11/exon 11 square in Fig. 8A. The complete absence of bi-allelic exon 11 mutations in the FANCD1/BRCA2 gene of FA-D1

patients supports the hypothesis of prenatal lethality of these particular and possibly other genotypes. Consistent with this notion, Rahman and Scott [143] observed an over-representation of BRCA2 mutations in exons 7 and 8, and the absence of homozygotes with the c.5946delT (also reported as c.6174delT) mutation [7] in exon 11 in FA-D1 patients, despite appreciable heterozygous population prevalence of the latter mutation in the Ashkenazim. Rahman and Scott [143] also pointed out that genotype–phenotype analyses of BRCA2 breast cancer pedigrees mirror-image the observations in biallelic BRCA2 mutation cases, with different cancer risks associated with mono-allelic truncating mutations in exon 11 when compared with mutations located upstream or downstream of this exon. The hypothesis of prenatal lethality of certain allelic combinations is also supported by the evidence that conventional Brca2−/− mice are non-viable, as are Brca1−/− mice [144]. With the exception of subtypes FA-D2, FA-I and, possibly, FA-D1, bi-allelic (or, in the case of FANCB, hemizygous) null mutations have been observed in patients of all other complementation groups. Unless there is evidence for an altered protein with some residual function, as has been shown for the exon 1 mutations (c.37C > T and c.67delG [7]) in FANCC [145], bi-allelic null mutations are generally expected to result in a severe phenotype. This canonical (but far from universal) association has been amply documented in subgroup FA-A, where complete loss of FANCA protein is associated with a severe phenotype. In contrast, FA-A patients with residual or altered protein display fewer somatic abnormalities, are somewhat older at onset of BMF, and have a lower risk of leukemia [2,3]. Using their novel multivariate phenotyping approach, Morales et al. [12] have recently confirmed that FA-A patients carrying

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disabling mutations on both alleles, which they postulate “to propagate throughout the protein” (meaning that these mutations exert their deleterious effects on the entire protein), are most severely affected. Mutations involving domains crucial for protein function such as the NLS, binding sites for interacting proteins, or sites of post-translational modifications are expected to result in severe abrogation of protein function. For proteins instrumental in the DNA damage response, intact cytoplasmic-nuclear shuttling appears crucial for function [146]. For example, virtually all diseasecausing mutations in the WRN gene prevent the nuclear localisation of the WRN helicase protein [147]. A similar situation may exist for mutations that affect cytoplasmic-nuclear shuttling of FANCA [148]. 4.3. Genotypes and other determinants compatible with long-term survival Which mutations are associated with the least severe phenotypes, and with the longest period of patient survival? In order to approach this question we have analyzed a cohort of adult FA patients (age 20 and older) [Neveling et al., in preparation]. Many of these patients have been subtyped, and mutation analysis has been completed in more than half of them. As shown in Fig. 9A, most of the adult patients were assigned to complementation group A. Given that subgroup A accounts for approximately 57% of all classified FA patients (cf. Fig. 1), the high prevalence of subtype FA-A among our adult patient cohort is not surprising. More impressive is the increase in the proportion of FA-A patients with age. They amount to 68% in the 20–29-year-old group, 71% in the group of 40–49 years, and 100% of >50-year-old FA patients (Fig. 9A). Unless most of the adult FA-A patients turn out to owe their relative longevity to somatic reversions in their blood stem cells (for which there is no evidence to date), these older patients must carry mutations that are compatible with long-term survival. What is the nature of these mutations? Most of the older FA-A patients are compound heterozygotes, with at least one allele carrying a missense mutation [Neveling et al., in preparation]. In many cases, these patients are recognized as FA only because of early cancers and/or unusually severe reactions to chemotherapy. A representative and highly instructive case belonging to this advanced age group is the patient described by Huck et al. [149]. Except for short stature (155 cm), this patient had no somatic abnormalities. She was diagnosed with leukopenia at age 23 and with thrombocytopenia at age 33, but did not require any blood product substitution. She developed breast cancer at age 37, tolerated 60 Gy of radiation therapy without major complications, but experienced a severe reaction to chemotherapy following diagnosis of contra-lateral breast cancer at age 45. At age 49, bone marrow biopsy revealed MDS with monosomy 7 which prompted additional cytogenetic and cell cycle studies, revealing an FA cellular phenotype. She turned out to be compound heterozygous for a nonsense and a missense mutation in FANCA (predicted to result in p.W410X and p.R517W [7]). Although FANCD2 monoubiquitination remained undetected in her cells by immunoblotting, cell survival and cell cycle studies were compatible with some residual activity of FANCA. One of the earliest examples of a mutation correlated with a mild clinical phenotype and frequent survival to adulthood is c.67delG (previously c.322delG) [7] in the first coding exon of FANCC [145]. Even though this mutation is predicted to result in a frameshift and an early premature stop codon, formally classifying it as a potentially severe change, Yamashita et al. [150] were able to explain the relatively mild phenotype associated with c.67delG [7] by the existence of a 50-kDa FANCC polypeptide reinitiated at methionine 55. Over-expression of this shorter FANCC polypeptide in an FANCC null cell line resulted in partial correction of MMC sensitivity, implying some degree of preserved FANCC function [150]. Among our cohort of FA patients surviving to adulthood, compound

Fig. 9. Characteristics of adult FA patients aged 20 years and older. Distributions of the four variables ‘complementation group’, ‘CAB score’, ‘gender’ and ‘history’ in the four age classes 20–29, 30–39, 40–49 and >50 years suggest factors that may influence long-term survival. The data include local observations (n = 92) and review of the literature (n = 42). Not all data were available for all patients. (A) The letters on top of the columns denote FA complementation groups (no. of patients included, n = 70). (B) The numbers on top of the columns denote congenital abnormality (CAB) scores [5] (no. of patients included, n = 56). (C) f and m on top of the columns denote female or male gender (no. of patients included, n = 134). (D) MOSAIC, MUT, HSCT and ANDR on top of the columns denote histories of mosaicism in the hematopoietic system, mild mutation, homologous stem cell transplantation or androgen therapy, respectively (no. of patients included, n = 99) (Neveling et al., in preparation).

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heterozygotes with c.67delG [7] make up the second most frequent group (cf. Fig. 9A, within group FA-C), and most of these patients lack severe malformations, are of average height, and have never required androgens, blood product substitution, or HSCT. Other factors associated with survival of FA patients to relatively old age are shown in Fig. 9B–D. Such predisposing factors include low to intermediate CAB scores (Fig. 9B), female gender (Fig. 9C), mosaicism in the hematological system, mild mutations, and HSCT (Fig. 9D). So far, our data are insufficient to resolve whether androgen therapy qualifies as one of these factors [Neveling et al., in preparation]. 4.4. Somatic reversions and “mild” mutations correlate with exceptional longevity Immunoblotting of FA-D2 patient cells illustrates how the presence of residual protein may correlate with long-term survival. Most FA-D2 patients are severely affected with high malformation scores and early onset BMF [137]. However, among 35 FA-D2 patients studied, eight patients survived to adulthood: three patients because of revertant mosaicism, and five patients because of apparently mild mutations. In contrast to three younger FA-D2 patients without any trace of FANCD2 protein (using standard Western blotting conditions, Fig. 10A, p3–p5), two of the patients with proven revertant mosaicism displayed near normal levels of both FANCD2 isoforms (Fig. 10A, p1 and p2). One of them (p1) was relatively severely affected (IUGR, microcephaly, mental retardation, renal disorder, hypogenitalism, radial ray hypoplasia) and developed MDS with death at age 21 from complications of HSCT. The other mosaic patient (p2) has a mild clinical phenotype and has normal blood cell counts at age 34, indicating early reversion of progenitor cells and/or stem cells, with successful repopulation of her bone marrow by the reverted cells [137]. In contrast to the three mosaic patients, FANCD2 protein levels in non-mosaic FA-D2 patients were much lower and could only be detected following over-exposure of X-ray films. As shown in Fig. 10B, the amount of residual protein in one of the exceptional

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long-lived FA-D2 patients (p10) clearly exceeded the levels of the other, non-mosaic FA-D2 patients. Relatively prominent FANCD2 bands of both isoforms were detected in this patient’s cells after prolonged exposure of the immunoblot to X-ray films, indicating a significant level and preserved function of the residual protein. At age 7, this patient displayed mild hypoplasia of the myeloid lineage but did not require blood product substitutions. At age 25, he presented with pigmentation anomalies, unilateral bifid thumb and hypogonadism, but otherwise had a close to normal phenotype. In addition to pigmentation anomalies, his younger brother had microphthalmia, a unilateral hypoplastic thumb, unilateral absence of the os metacarpale I, and glandular hypospadia. At age 20, his blood cell counts were in the low normal range. The brothers were homozygous for the missense change c.2444G > A (p.R815Q) [7], thus qualifying as a prototypic example of a mild mutation in FANCD2. These examples of rare, long-lived FA-D2 patients illustrate how the clinical course of FA may be influenced by the emergence of revertant mosaicism or the presence of a “mild” mutation (as defined by the presence of functionally intact residual protein and an attenuated phenotype). Whereas the emergence of revertant mosaicism is difficult to predict and its frequency varies among the different FA complementation groups [125], criteria for the presence of a “mild” mutation can be more readily defined as any type of genetic alteration that results in a less severe clinical course. The example of FANCD2 shows that “mild” must not be confused with “hypomorphic”. Many mutations may be hypomorphic so that they preserve some degree of residual protein with at least partial function, but this may not necessarily result in a less severe phenotype and a milder disease course. Unless more is known about the structure and function of the individual FA proteins and of putative suppressor alleles at other loci, it remains difficult to predict the phenotypic consequences of mutations that result in variable amounts of or in structurally altered proteins. How knowledge of protein structure facilitates the prediction of functional consequences is illustrated by the mutation p.R371W [7] in FANCE. As a typical missense change, one might predict relatively mild phenotypic consequences of this alteration. However, Nookala et al. [151] were able to show that the substitution of arginine with trypthophane by the c.1111C > T base change [7] results in the loss of several structurally important hydrogen bonds in one of the FANC repeats of FANCE. Loss of these hydrogen bonds affects the ternary structure of the FANCE protein and leads to its destabilization. This results in impaired function [151]. 4.5. Genetic alterations specifically associated with neoplasia

Fig. 10. FANCD2 immunoblotting of lymphoblast extracts from FA-D2 patients. (A) Lymphoblast cell lines from two FA-D2 patients with proven revertant mosaicism (p1 and p2, lanes 3 and 4) show near normal levels of both FANCD2 isoforms compared with those of a normal control (CON, lane 1). An FA-A control (FA-A, lane 2) reveals a single FANCD2-S band. In contrast, cell lines from FA-D2 patients p3–p5 without mosaicism fail to show either band (lanes 5–7) on standard exposure conditions. (B) Using over-exposure of X-ray films as evidenced by the unusually dark signals from two normal controls (CON, lanes 1 and 7), cell lines from five non-mosaic FA-D2 patients (p6–p10) show both FANCD2 bands faintly (lanes 2–6), indicating residual functional protein. Residual protein levels vary among the different FA-D2 cell lines tested. The cell line from patient 10 with mild clinical phenotype, homozygous for a less penetrating mutation, reveals relatively prominent D2 bands (p10, line 6). All cultures were exposed to 50 ng/ml MMC for 14 h.

As already noted, close to 100% of FA-D1 patients develop neoplasias by the age of 5 years [124]. The same situation exists for FA-N patients, where not a single child is free of neoplasia or remained alive beyond that age [57]. Of 23 FA-D1 patients, nine developed AML within the first 3 years of life [152]. Among these, 5 children carried the c.631 + 2T > G (IVS7 + 2T > G) splice site mutation [7] of FANCD1/BRCA2 in one or both alleles, leading to the suggestion that this particular type of alteration may predispose to AML. If so, this predisposition exists only in the context of the FA-D1 genotype, since no such c.631 + 2T > G (IVS7 + 2T > G) change [7] was detected among 190 non-FA childhood leukemias [152]. Brain tumors (medulloblastomas) arising in FA-D1 patients were predominantly, but not exclusively associated with the alterations c.658 659delGT (c.886 887delGT) and c.5946delT (c.6174delT) [7], but overall numbers are small. These putative associations therefore require confirmation in larger patient cohorts [124]. Except for mutations in FANCD1 and FANCN, there are no consistent genotype–phenotype correlations with respect to neoplasia. There is a tendency for over-representation of carriers of large deletions (such as EX12 31del [7]) in the FANCA gene among FA chil-

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dren with AML [2], but large deletions involving exons 12–31 and others are abundant in FANCA. More data are therefore required. Initial expectations that alterations of FA genes would frequently be found in FA-typical neoplasms arising in the general population have not been substantiated. For example, among 107 non-FA children affected by AML only 2 were found to carry bi-allelic mutations in an FA gene (FANCG). This observation merely confirms that FA presenting as primary AML might not have previously been diagnosed [153]. Failure to recognize the underlying condition may have fatal consequences, as these patients may experience severe toxicity when exposed to conventional chemotherapy [154]. Cleton-Jansen et al. [155] failed to detect any FANCA mutations in 19 breast tumors. Surprisingly, FA genes appear to be affected in only a small proportion of neoplasms arising in non-FA patients. This suggests elusive selective advantage for neoplastic cells carrying such mutations [72,156]. The only known exception to this generalization is the epigenetic silencing of FANCF in various kinds of tumors. However, silencing of FANCF may represent a mere bystander effect rather than a primary effect, since FANCF is located in a known imprinting hotspot of the human genome [156]. In addition to the striking correlations between genetic alterations and the emergence of neoplasia in case of FANCD1 and FANCN gene mutations, monosomy 7 or partial trisomies or tetrasomies of the chromosomal region 3q26–29 arising in preleukemic bone marrow cells of FA patients may represent the best example to date for a genotype–phenotype correlation with respect to the emergence of MDS and/or AML [41,157]. As noted above, amplification and rearrangements of 3q result in over-expression of EVI1. This correlation has also been observed in an FA-derived AML cell line with bi-allelic mutations in FANCD1 [42]. 3q aberrations and high EVI1 expression levels predict adverse outcome of AML, regardless of whether the leukemia arises on an FA or on a non-FA background [158].

5. Fanconi anemia as a segmental progeroid entity 5.1. Cellular phenotype as driving force of the FA progeroid features As stated by Liu et al. more than 10 years ago, a number of features of the FA phenotype is reminiscent of cellular senescence [159]. For example, in normal ageing, the rate of chromosome and telomere lesions increases as a function of age, both in vivo and ex vivo [160]. There is a steady increase of lymphocytes arrested in the G2 phase of the cell cycle when activated in culture, likewise as a function of donor age [91]. Genetic instability has been recognized as a common denominator of the classical human progeroid syndromes [161]. There is compelling evidence from animal models that DNA repair defects impair stem cell functions and promote ageing [162]. Hypocellularity of the bone marrow is a consistent feature of advanced age, but cytopenia progresses much more rapidly in FA than in normative ageing. Nevertheless, many of the clinical features of FA, including BMF, can be interpreted as manifestations of premature ageing at the cellular, organ and organismal levels. Unrepaired or misrepaired DNA damage leads to cell loss, either by increased cell death rates or by cell cycle arrest, or results in cellular senescence, which also depletes the number of proliferating cells. The first and foremost manifestations of disturbed proliferative homeostasis are intrauterine and postnatal growth retardation which affect at least 70% of FA patients. The majority of FA children and FA adults are underweight and display clinical evidence for sarcopenia. Over-production of TNF-␣ is a consistent feature of FA [111,114,163] and may contribute to reduced muscle mass in FA patients, as it does in normative ageing [164]. TNF-␣ production and TNF-␣-induced apoptosis are increased during physiological ageing [165]. Among the many pleiotropic actions of TNF-␣ that may

relate to the premature ageing phenotype in FA are suppression of erythropoiesis, hypersensitivity to apoptotic cytokine cues, and insulin resistance [111,164]. Even the increased risks of leukemic clonal evolution and AML have been attributed to TNF-␣ mediated induction of ROS, increasing the genetic instability in hematopoietic precursor cells [166]. As pointed out by D’Andrea, FA cells are highly sensitive to ROS not because they are unable to detoxify ROS, but because they are unable to respond properly to ROS-mediated DNA damage [167]. Among likely clinical manifestations of genetic instability are pigmentation changes as they commonly occur during normal ageing. These pigment alterations are much more frequent and prominent in disorders prone to benign or malignant proliferations of variable cell origin, including FA [168]. In the case of neurofibromatosis (NF1)-associated café-au-lait macules (CALMs), a second hit in the NF1 gene has been documented in the affected skin area, indicating somatic mutation [169,170]. No such data are available for FA-associated CALMs, but these lesions clearly represent foci of disturbed proliferative homeostasis which is a hallmark of ageingrelated skin changes [171]. Endocrine disorders, many of which invariably occur during normative ageing, are present in 70–80% of FA patients at comparatively young ages [172,173]. These include growth hormone deficiency, hypothyroidism and hypogonadism. Some of these hormonal deficits may be associated with structural defects, such as central nervous system malformations, e.g. midline brain defects [173], small pituitary size [174] or pituitary stalk interruption syndrome [175]. However, the majority of FA patients exhibit endocrine dysfunction in the absence of gross structural deficits. In one study of 45 FA patients aged 2–49 years [173], 65% of peripubertal or postpubertal patients had gonadal dysfunction. Premature ovarian failure [176–178] which can be interpreted as a manifestation of accelerated ageing of the hypothalamic–pituitary axis, is the rule in postpubertal FA females, and fertility is severely reduced in FA males due to impaired spermatogenesis [175]. Another prominent feature reminiscent of premature ageing is the high frequency of osteopenia and osteoporosis, affecting 92% of FA patients older than 18 [173]. In addition, FA patients frequently exhibit abnormalities of glucose metabolism, including insulin resistance, hyperglycemia, glucose intolerance and overt diabetes mellitus [173]. The MDS represents a disorder of the hematopoietic system and is classified as a preleukemic condition. Its incidence increases with age, with the rate of affected individuals rising dramatically after age 70, and with a median age at diagnosis of approximately 71 years. MDS represents a major health problem in the elderly population [179]. FA is associated with a high risk for MDS; its cumulative incidence amounts to almost 7% [180]. However, the median age at diagnosis of MDS in FA is 9.3 years, more than 60 years earlier than MDS in the general population. This suggests a short cut for the manifestation of a disorder that typically arises in the elderly. A similar reduction in time applies to the manifestation of SCCs of the oral cavity and the anogenital region, which in nonFA patients are typical tumors of advanced age. These tumors occur at much younger ages in FA patients, and without conclusive evidence for HPV infection in the case of anogenital tumors [181]. Due to conditioning procedures and chronic graft-versus-host disease (GVHD), SCCs are more frequent after HSCT, but there are a number of FA patients who develop SCC prior to or without any HSCT [182]. Mild to moderate immunodeficiency is a hallmark of ageing, possibly explaining, at least in part, the age-related increase of neoplastic cell growth [183,184]. There is some evidence for dysfunctions of the immune system in FA which may contribute to early and frequent tumorigenesis [185]. SCCs preferentially develop at sites where squamous epithelia are exposed to ambient oxygen concentrations. Since most FA patients neither smoke nor drink,

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5.2. Speculation: FA—a model of premature onset and accelerated progression of ageing

Fig. 11. Segmental progeroid features in FA. Portrayed is a 28-year-old patient of complementation group FA-A (compound heterozygous for c.2222 + 1G > T and EX32del in FANCA) [7]. His progeroid symptoms include hearing impairment, cataract, sicca syndrome, alopecia, actinic keratosis and SCC. See text for details. Courtesy of Ralf Dietrich.

The impressive progeroid features of FA can be viewed as the most compelling summarization of its range of genotype–phenotype correlations. In that sense, FA can be seen as a range of tissue-specific premature onset and accelerated ageing. All of the FA proteins undoubtedly contribute, within multiple pathways, networks, and interactions, to the maintenance of genomic stability. At the cellular level, impairment of FA gene functions leads to a surprisingly uniform susceptibility to the adverse effects of specific types of genotoxic agents including ROS. Given the unique sensitivity of FA cells to ROS, FA may represent the only human model of the free radical theory of ageing. A defective DNA damage response results in impaired proliferation, increased cell death, and increased numbers of genetically altered cells, which in the presence of a compromised immune system may promote carcinogenesis. Several model systems have contributed to our understanding how ageing may be driven by DNA damage [187–190]. If one defines ageing as increasing loss of tissue homeostasis [191] – or the ability of an organ to recover to normal function after stress – some of the symptoms of the pleiotropic FA phenotype fit with this definition and can be viewed as the result of an accelerated loss of homeostasis. As such, FA may represent a compression in time of the natural history and physiologic pleiotropy of the normal ageing process. Conflict of interest The authors declare that there are no conflicts of interest.

the genetically determined susceptibility of FA cells to ROS might bear a part in their high risk for malignant transformation of squamous cells. A similar pathogenic mechanism may account for the emergence of basal cell carcinomas and actinic skin lesions, which prematurely and frequently emerge in adult FA patients. In order to illustrate some aspects of the FA phenotype that is reminiscent of premature ageing, Fig. 11 portrays a 28-year-old FA-A male with bilateral cataracts and sicca syndrome with opacities and neovascularization of his both corneas, resulting in almost complete blindness. Skin changes include alopecia and actinic keratosis-like foci with disseminated inflammatory eruptions and atrophical lesions as a manifestation of chronic GVHD after matched sibling HSCT at age 9. Hundreds of his skin lesions were treated with photodynamic therapy (using 5-aminolaevulinic acid as photosensitizer and 630 nm red-laser ablation) within the past 5 years in order to minimize the risk for the emergence of multiple SCCs in situ. A spino-cellular carcinoma was surgically removed from his lower lip 3 years ago, followed by neck dissection and radiotherapy. The patient is dependent on hearing aids because of inborn partial deafness. Other FA-typical clinical features include small stature, microcephaly and a hypoplastic left thumb. Males generally experience higher morbidity and mortality for most age-related and other disorders [186]. The high morbidity of FA patients results in increased death rates throughout lifetime, leading to sharply reduced life expectancy in both genders. As in the general population, FA males statistically die earlier than FA females. In our adult FA patient cohort with most individuals belonging to the 20–40 year range (cf. Section 4.3), we observed nearly twice as many females as males. In the general population, a 2:1 ratio in favor of females (as noted in our adult FA cohort) is reached no sooner than age 85, and a 3:1 ratio in centenarians [186]. Again, the observed premature gender difference is compatible with the notion that FA patients experience a time lapse compression of what may represent a physiological age-related gender difference in our species.

Acknowledgments We are indebted to Richard Friedl, Gitta Emmert and Birgit Gottwald for outstanding technical support. We gratefully acknowledge help with data collection and processing by Grit Hohnbaum, and the provision of retroviral vector techniques by Helmut Hanenberg. Special thanks go to George M. Martin for continuing encouragement and support. This review would not have been possible without myriad contributions from FA patients and their relatives and physicians over many years. Specifically, we thank the family support groups, Deutsche Fanconi Anaemie Hilfe, Aktionskreis Fanconi Anaemie, and the Fanconi Anemia Research Fund for facilitating informative exchange and providing financial support. References [1] P.F. Giampietro, P.C. Verlander, J.G. Davis, A.D. Auerbach, Diagnosis of Fanconi anemia in patients without congenital malformations: an international Fanconi Anemia Registry Study, Am. J. Med. Genet. 68 (1997) 58–61. [2] L. Faivre, P. Guardiola, C. Lewis, I. Dokal, W. Ebell, A. Zatterale, C. Altay, J. Poole, D. Stones, M.L. Kwee, M. van Weel-Sipman, C. Havenga, N. Morgan, J. de Winter, M. Digweed, A. Savoia, J. Pronk, T. de Ravel, S. Jansen, H. Joenje, E. Gluckman, C.G. Mathew, Association of complementation group and mutation type with clinical outcome in Fanconi anemia. European Fanconi Anemia Research Group, Blood 96 (2000) 4064–4070. [3] D.I. Kutler, B. Singh, J. Satagopan, S.D. Batish, M. Berwick, P.F. Giampietro, H. Hanenberg, A.D. Auerbach, A 20-year perspective on the International Fanconi Anemia Registry (IFAR), Blood 101 (2003) 1249–1256. [4] B.P. Alter, The association between FANCD1/BRCA2 mutations and leukaemia, Br. J. Haematol. 133 (2006) 446–448, author reply 448. [5] P.S. Rosenberg, Y. Huang, B.P. Alter, Individualized risks of first adverse events in patients with Fanconi anemia, Blood 104 (2004) 350–355. [6] P.S. Rosenberg, B.P. Alter, W. Ebell, Cancer risks in Fanconi anemia: findings from the German Fanconi Anemia Registry, Haematologica 93 (2008) 511–517. [7] Human genome variation society. Nomenclature for the description of sequence variations, http://www.hgvs.org/mutnomen/. [8] D.I. Kutler, A.D. Auerbach, Fanconi anemia in Ashkenazi Jews, Fam. Cancer 3 (2004) 241–248.

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