Best Practice & Research Clinical Haematology Vol. 14, No. 3, pp. 631±644, 2001

doi:10.1053/beha.2001.0158, available online at http://www.idealibrary.com on

9 Chromosome instability syndromes A. M. R. Taylor

PhD

Professor of Cancer Genetics CRC Institute for Cancer studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, UK

The chromosome instability syndromes, ataxia telangiectasia (A-T), Fanconi anaemia (FA) and Bloom syndrome (BS) have been known for many years. More recently Nijmegen breakage syndrome (NBS) and ataxia telangiectasia-like disorder (ATLD) have been identi®ed. A-T, ATLD and NBS form a group of disorders all of which show very similar cellular features that result from the consequences of increased sensitivity to ionizing radiation (IR). They also share some clinical features, particularly A-T and ATLD, and all show an immunode®ciency. A-T and NBS both show a predisposition to lymphoid tumours. Fanconi anaemia can be caused by mutations in eight di€erent genes, although the majority of mutations are accounted for by FANCA and FANCC. The very rare Bloom syndrome is caused by mutation in a single gene, BLM. An important feature which all of these disorders have in common is that the genes identi®ed are involved in aspects of recombination repair of DNA damage. Key words: ataxia telangiectasia; ataxia telangiectasa-like disorder; Nijmegen breakage syndrome; Fanconi anaemia; Bloom syndrome; chromosome instability; cancer.

The commonly acknowledged chromosome instability syndromes are ataxia telangiectasia (A-T), Fanconi anaemia (FA) and Bloom syndrome (BS). The characteristic shared by all of these disorders is a high level of spontaneously occurring chromosome abnormalities although, importantly, the type of abnormality is di€erent in each syndrome. Similarly, each disorder shows a higher than normal frequency of induced chromosome abnormalities, although again, for each disorder the inducing agent is di€erent. Each syndrome also has its own individual spectrum of tumours±mainly lymphoid tumours in A-T, myeloid leukaemia and epithelial cell tumours in FA and a wide range of epithelial tumours and leukaemias in BS. Bloom syndrome is caused by a single gene, BLM; A-T is caused by mutation in the ATM gene, but also shows locus heterogeneity because ataxia telangiectasia-like disorder (ATLD) is caused by mutation in the hMRE11 gene. A very similar disorder at the cellular level, Nijmegen breakage syndrome (NBS), is caused by mutation in the NBS1 gene. Fanconi anaemia is now known to be caused by at least eight di€erent genes of which six, FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG have been cloned. 1521±6926/01/030631‡14 $35.00/00

c 2001 Harcourt Publishers Ltd. *

632 A. M. R. Taylor

ATAXIA TELANGIECTASIA, ATAXIA TELANGIECTASIA-LIKE DISORDER AND NIJMEGEN BREAKAGE SYNDROME Ataxia telangiectasia Ataxia telangiectasia is a recessively inherited disorder. The main clinical features are a progressive cerebellar degeneration resulting in upper and lower limb ataxia, speech diculties and abnormal eye movements. The telangiectasia is most obvious on the bulbar conjunctiva. Immunode®ciency can be demonstrated as a laboratory feature in most patients, although only in a proportion of patients is this manifest as an increased susceptibility to infection. Patients are unusually sensitive to ionizing radiation; this becomes a very important factor when radiotherapy is considered as a suitable treatment for tumours in these patients because exposure of A-T patients to therapeutic doses of ionizing radiation has proved to be fatal.1 Chromosome instability in A-T The most characteristic chromosome abnormality of A-T is the translocation chromosome involving breakpoints within the T cell receptor genes in peripheral T cells. Translocations may occur between any of the T cell receptor genes on chromosome 14q11 (TCRa and d), 7q14 (TCRg) and 7q35 (TCRb) to give a variety of translocation chromosomes. These include t(7;14)(q35;q11), t(7;14)(p13;q11), t(7;7)(p13;q35) and inv(7)(p13q35).2 The characteristic of these translocation-carrying cells is that they have some proliferative capacity, although for most this is limited. Typically, approximately 10% of phytohaemagglutinin (PHA)-stimulated T cells from A-T patients carry such translocations. Normal individuals also carry these translocations but at the much lower rate of about 1/500 cells.3 Three translocations however, t(14;14)(q11;q32) inv(14)(q11q32) and t(X;14)(q28;q11), involving translocation of the TCRa gene to either of two oncogenes, TCL1 (at 14q32)4 or MTCP1 (at Xq28)5±7, result in a much greater proliferative capacity of these cells.2 These clones may occupy up to 90% of the PHA-stimulated T cell compartment. An interesting feature of these large translocation clones is the formation of telomeric fusion chromosomes in a proportion of the cells, probably as a result of the loss of telomere sequence.8 These translocations alone are not sucient for tumour transformation because the clone cells may exist for many years in the absence of any tumour. The same cells, however, undergo additional genetic change, resulting in the development of T cell prolymphocytic leukaemia (T-PLL).2 A feature of the tumour cells is the large number of observable genetic changes that have occurred. The same initial cytogenetic translocation and the same T cell receptor gene rearrangement are seen in the pre-leukaemic and subsequent leukaemic cells from these patients, indicating the crucial role of this translocation in the tumour process.9 In ataxia telangiectasia, therefore, there is evidence for a clear link between chromosome instability and the development of lymphoid tumours.2 A pre-existing large clone in the peripheral blood lymphocytes of A-T patients, therefore, is associated with a high risk for the development of T-PLL, and these patients should be examined regularly. Another important feature of cultured A-T cells is their increased sensitivity to ionizing radiation.10,11 Following exposure of cells to 1-2 Gray X-rays at the G2 phase of the cell cycle, classical A-T cells typically show approximately 10 times the level of induced-chromatid-type damage compared with normal cells.11 Irradiation at G0 also produces increased levels of unrepaired chromosome damage in A-T cells.11 Chromosome instability can also be demonstrated in the B lymphocytes of A-T patients, and the translocations involve breakpoints in the immunoglobulin genes. For

Chromosome instability syndromes 633

example, a t(2;14)(p11;q32) translocation involving a breakpoint within IgH and on 2p11 outside and proximal to IgK has been reported.12,13 A-T patients can carry translocation clones in both B and T cell compartments.2 The ATM de®ciency, therefore, a€ects rearrangement of both T cell receptor and immunoglobulin genes but it is not known whether translocations carrying B lymphocytes can give rise to tumours in A-T patients. Chromosome translocations involving immune system genes are not present in cultured skin ®broblasts from A-T patients. Tumours in A-T The majority of tumours in A-T patients have a lymphoid origin and include Hodgkin's disease, B cell non-Hodgkin's lymphoma, T cell lymphoma, T cell acute lymphoblastic leukaemia and T-PLL. Apart from T-PLL it is not known whether there is a direct role for chromosome translocation in the development of the other tumours. Since the cloning of the ATM gene14, analysis of patients with lymphoid tumours has shown that mutation at a single location is not associated with predisposition to leukaemia. Mutations are scattered across the whole of the cDNA in these patients. Interestingly, in families with two or more siblings with A-T, the same histological type of lymphoid tumour is observed. ATM gene mutations in sporadic tumours It has been shown that the ATM gene is mutated in sporadic T cell prolymphocytic leukaemia (T-PLL) tumours15±17 in B cell chronic lymphocytic leukaemia18±20 and in mantle-cell lymphoma21 and probably, therefore, has a role in the development of sporadic lymphoid tumours. The precise mechanism of the involvement of ATM may di€er between di€erent types of tumour. Sporadic cases of T-PLL, like those in A-T patients, also often have translocations involving inv(14), t(14;14) or t(X;14) in the tumour cells.2,6 In these cases the history of the development of the tumour clone is not known. Loss of normal-functioning ATM, however, is implied from the ®nding that a combination of loss of heterozygosity in the region of the ATM gene in one allele and an ATM missense mutation, mostly in the C terminal region where the catalytic domain is situated in the second allele, is a frequent ®nding.15 In contrast, B-CLL tumours may have di€erent combinations of allelic changes leading to either complete loss of ATM or compromised function of ATM.18,20 Bearing in mind the tendency for A-T B lymphocytes to produce chromosome translocations involving immunoglobulin genes, B-CLL tumours with compromised ATM show no evidence of such chromosomal translocations. Mantle-cell lymphoma, however, associated with the t(11;14) translocations21, does show the presence of ATM mutations. ATM function ATM is principally a nuclear protein. Most work on the functions of ATM has concentrated on its role as a serine/threonine protein kinase that is activated by exposure of cells to ionizing radiation22,23 and it has many substrates, including BRCA1, all with a general consensus motif (SQ/TQ).24 A major role of ATM is to regulate the p53 responses which result in G1 arrest after exposure of the cell to some forms of DNA damage. Another important checkpoint failure in A-T cells occurs in S phase and gives rise to the distinctive phenotype of radioresistant DNA synthesis±failure of cells actually in S phase to inhibit DNA synthesis following exposure of cells to ionizing

634 A. M. R. Taylor

radiation (IR). The defective ability of A-T cells to arrest at the G2/M checkpoint also suggests a direct role for ATM in this process. Early work demonstrated the failure of cellular repair systems in A-T.25 However, it has not been possible to demonstrate a substantial defect in DNA double strand breaks (DSB) repair. Probably 95% of radiation-induced DNA DSB are repaired in an ATM-independent manner. Defects in intrachromosomal recombination pathways have been reported in A-T cells26 and also cells from Atmÿ/ÿmice.27 There are also several ®ndings implicating ATM function in homologous recombination. Recently it has been reported that radiation-induced assembly of Rad51 and Rad52 components of the recombination complex requires ATM.28 In the DT40 chicken cell system, defects in both HRR (homologous recombination repair) and NHEJ (non-homologous end-joining) genes have been implicated in the production of chromosome abnormalities. A combination of ATMÿ/ÿ and KU70ÿ/ÿ increases the chromosomal instability phenotype of ATMÿ/ÿ DT40 cells. In contrast, RAD54ÿ/ÿ ATMÿ/ÿ does not increase chromosomal instability, suggesting that ATM might function in a homologous recombination repair pathway of DNA repair.29 Atm null mouse The atmÿ/ÿ mouse has many of the feature of ataxia telangiectasia, although these animals die at about 4 months of age with T cell lymphoma. The tumours also show the presence of translocations involving breakage within the T cell receptor genes. There is no cerebellar ataxia, although ccrebellar defects can be demonstrated.30±32 Ataxia telangiectasia-like disorder (ATLD) A small proportion of patients with ataxia telangiectasia have mutations in the hMRE11 gene rather than in ATM. This disorder has been called ataxia telangiectasia-like disorder (ATLD).33 The clinical features of ATLD are largely indistinguishable from those of A-T, although the ATLD patients do not have telangiectasia. The T lymphocytes of ATLD patients, like those of A-T patients, show an increased level of translocations of chromosomes 7 and 14 involving immune system genes, but nothing is known about chromosomal abnormalities in B lymphocytes in these patients. T lymphocytes and cultured skin ®broblasts from ATLD patients also show the same increased chromosomal radiosensitivity compared with normal cells. So far no tumours have been reported in these patients. An e€ectively null hMRE11 allele, expressing no protein, can occur through the mechanism of nonsense-mediated mRNA decay whereby, in the presence of particular mutations, the newly synthesized mRNA is degraded and essentially no transcript is available from the allele.34 This mechanism of loss of gene function has not been reported for the ATM gene. hMRE11, however, is an essential gene35 so that, in the recessive disorder ATLD, some residual function of hMRE11 is necessary. Typically the second allele may be hypomorphic, that is, having an imperfect function compared with a normal allele, such as a partially functional protein resulting from a missense mutation or a truncated protein with some residual function.33,34 Nijmegen breakage syndrome Nijmegen breakage syndrome (NBS)36 is the third disorder in which there are translocations involving chromosomes 7 and 14 in peripheral T cells. The average

Chromosome instability syndromes 635

frequency of translocations may be greater than in A-T patients. As with A-T patients, large translocation clones may be present in occasional patients, again showing telomeric fusions. There is also an increase in chromosomal radiosensitivity in these patients. NBS is prevalent among those of Eastern and Central European descent, and the majority of patients carry the NBS1 657del5 founder mutation, a truncating mutation which causes premature termination at codon 219.37 Homozygous null mutations of Nbs1, however, are lethal in mice.38 In cells from NBS patients with the 657del5 mutation a 70 kDa protein lacking the N terminal region of the protein is produced by internal translation initiation within the NBS1 mRNA using an open reading frame generated by the 657del5 frameshift. The 70 kDa protein is associated with the Mre11 complex and may result in partial functioning of the complex resulting in the NBS phenotype.39 A high proportion of NBS patients develop lymphoid malignancies but, in contrast to A-T, the majority of these are of B cell origin, although some T cell tumours are reported.40 A-T, ATLD, NBS and interactions of ATM, hMre11 and Nbs1 Why do A-T (ATM), ATLD (hMRE11) and NBS (NBS1) patients' cells all have similar chromosome abnormalities in the peripheral circulation and a similar chromosomal hypersensitivity to ionizing radiation? The Nbs1/hMre11 proteins together with hRad50 form a complex which has a role in DNA double-strand break repair. Even though hMre11 and Nbs1 are part of a single complex, the clinical features of ATLD are more like A-T than NBS. The fact that hMRE11 mutation can produce an A-T like phenotype links the ATM with the hMre11 complex. Cells from all three groups of patients have one other important feature in common which is an inability to trigger an S phase checkpoint following damage by ionizing radiation. ATM phosphorylates Nbs1 in vivo on ser-343 and this phosphorylation event is required for the IR-induced S phase checkpoint.41,42 It is likely that ATM and the components of the hMre11 complex function together as an S-phase checkpoint complex. The S phase checkpoint may also involve elements of the homologous recombination repair (HRR) process because BRCA1, for example, complexes with hMre11. FANCONI ANAEMIA Fanconi anaemia is a recessive disorder characterized by multiple congenital abnormalities, bone marrow failure and a predisposition to di€erent types of tumour, including acute myeloblastic leukaemia and di€erent epithelial cell tumours.43 A small proportion of FA patients, however, have no congenital abnormalities44, and it is recognized as a clinically heterogeneous disorder. Chromosome instability in Fanconi anaemia At the chromosome level chromatid breaks can be increased in number in stimulated T cells from FA patients, although this response can be variable. This is di€erent from A-T cells, for example, where the level of spontaneous breaks in peripheral T cells is very similar to that in normal cells. The striking cytogenetic feature of FA is the unusual sensitivity to DNA cross-linking agents such as mitomycin C and diepoxybutane, which is manifested as an increased level of chromatid-type damage, including triradials and quadriradials and more complex interchanges.45

636 A. M. R. Taylor

Genes for Fanconi anaemia The heterogeneity seen at the clinical level in FA is re¯ected at the genetic level where complementation studies have suggested eight di€erent genes for Fanconi anaemia of which FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG have been cloned.46±52 FANCA and FANCC make up the majority of cases. The FA genes are quite di€erent from each other, showing little or no homology. The presumption was that the FA genes act in the resolution of DNA cross-links. Because defects in all of these genes cause features of FA, the expectation is that they will be shown to be in a common pathway, or a€ecting a pathway in common, although, at present, the precise function of the pathway involving these di€erent proteins is unresolved. Physical complexes of various combinations of these proteins have been demonstrated in normal cells.53±55 The FANCA, C and G proteins normally form a complex localized primarily to the nucleus but this complex formation is not observed in cells from FANCA, B, C, E, F and G, suggesting that a component upstream of these proteins is important in their stabilization.56,57 In contrast, the complex is seen in FANCD2 cells (PD20 cell line) and in the presumed FANCD1 (HSC62 cell line) suggesting that the FANCD1 and FANCD2 products function downstream of the FA protein complex.58 Recently it has been shown that a nuclear complex containing FANCA, FANCC, FANCF and FANCG proteins is required for activation of the FANCD2 protein.58 Normal lymphoblastoid cell lines express two forms of FANCD2 protein, a short (FANCD2-S 155 kDa) and a long form (FANCD2-L, 162 kDa). The 155 kDa protein is the primary translation product of the cloned cDNA. FA lines from complementation groups A, B, C, E, G and F expressed only FANCD2-S. Functional correction of the cell lines with corresponding cDNA restored the protein complex and expression of FANCD2-L. It was shown that the FA protein complex containing FANCA, FANCC, FANCF and FANCG regulates expression of the two isoforms of FANCD2. These six FA genes therefore interact in a common pathway. The FANCD2-L results from mono-ubiquitination and not phosphorylation of FANCD2-S, and the FA protein complex is required for this modi®cation of FANCD2.58 One of the consequences of damage to a cell is the relocalization of various damageresponse proteins to foci of activity, presumably at the site of damage. Formation of nuclear foci containing FANCD2 requires an intact FA pathway, and mitomycin C activates conversion of FAND2-S to FANCD2-L in normal cells. An increase in FANCD2-L was paralleled by an increase in FANCD2 foci. Ionizing radiation and exposure to UV light can also induce FANCD2 focus formation in HeLa cells. FA cells from di€erent complementation groups, however, failed to activate FANCD2-L and failed to activate FANCD2 foci in response to mytomycin (MMC) and ionizing radiation, suggesting that sensitivity of FA cells to MMC or IR results in part from failure to activate FANCD2-L nuclear foci.58 BRCA1 co-localizes in ionizing-radiation-inducible foci with other proteins such as Rad51 or the Mre11/Rad50 complex. Cells with bi-allelic mutations in BRCA1 have a defect in DNA repair and are sensitive to agents such as IR and MMC. One of the most interesting recent ®ndings on the biology of Fanconi anaemia cells is the observation that radiation-induced foci contained both BRCA1 and FANCD2. These two proteins also co-localized in foci during S phase, and this interaction of FANCD2 and BRCA1 was con®rmed by co-immunoprecipitation. The suggestion, therefore, is of a functional interaction of BRCA1 and FANCD2.58 These interesting data can be summarized as showing that FANCD2 associates with recombination proteins. It is suggested that co-localization of FANCD2 with Rad51 and

Chromosome instability syndromes 637

RPA at recombination nodules early in meiosis I places FAND2 at possible sites of DNA DSB and suggests that FANCD2 may be part of the process of homologous recombination.58 It is now possible to link the function of all FA proteins through FANCD2 to other proteins known to be involved in homologous recombination repair (HRR). FA null mice The FANCC null mouse showed no developmental or haematological abnormalities, although spleen cells showed damage in response to exposure to mitomycin C or diepoxybutane. In both male and female mice there were reduced numbers of germ cells, resulting in impaired fertility.59,60 The colony-forming capacity of marrow progenitor cells was abnormal in vitro and, interestingly, progenitor cells were unusually sensitive to interferon gamma (IFN-g). This result suggested that FANCC protein plays a role in growth, di€erentiation or survival of progenitor cells by suppressing an IFN-g mediated apoptotic pathway.60 This ®nding has stimulated subsequent work on this pathway in Fanconi patient cells. Mice de®cient in FANCA have also been generated. They are viable and have no detectable developmental abnormalities. Cells from these animals were unusually sensitive to mitomycin C, con®rming their FA-like cellular phenotype. As with FANCC mice, both male and female mice showed hypogonadism and impaired fertility.61 FA proteins in haematopoietic progenitor cells and in sporadic acute myeloid tumours The IFN-g induced apoptotic response in haematopoietic stem cells from an FANCC patient occurred at a much lower IFN dose compared with stem cells from a normal individual. The normal FANC protein therefore appears to modulate IFN-g signals preventing an apoptotic response.62 The high risk of developing AML in FA suggests a normal protective role for the FA protein complex. The protein products of FA genes were compared in established AML lines and also in primary AML tumour cells. Aberrant protein pro®les were observed in 5/10 lines and 1/15 primary AML. The conclusion from this work was that some disturbance of the `FA pathway' may play a part in the development of sporadic AML.63 Bone marrow transplantation in Fanconi anaemia Without treatment the bone marrow failure in FA is usually fatal before the age of 20 years and there is a predisposition to myelodysplasia.64 Allogeneic stem cell transplantation is the only way to restore normal haematopoiesis in FA.65 A major problem is the conditioning regime for transplantation. A dose reduction of the alkylating agents used for conditioning is necessary because of the increased sensitivity of FA cells to DNA cross-linking agents. In a retrospective study analysing the outcome of 69 allogeneic stem cell transplantations using HLA-matched unrelated donors, the 3-year probability of survival was 33%.65 The study identi®ed a correlation between the clinical phenotype of the FA patient and clinical outcome in that the presence of congenital malformations was correlated with poorer survival.65 The presence of malformations was associated with a higher incidence of acute graft-versus-host disease (GVHD). In addition, positive recipient cytomegalovirus serology, the use of androgens before transplant and female donors were associated with a worse outcome.65 The survival rate of 33% compared with a greater than 70% probability of survival reported if HLA-matched sibling donors were used.66±68

638 A. M. R. Taylor

The role of radiation treatment in the conditioning regimen for transplantation has been questioned. Conditioning with low-dose cyclophosphamide and thoracoabdominal irradiation is common, although not all centres advocate the use of radiation.67,68 A minority of FA patients may have an unusual radiosensitivity45, another indicator of the heterogeneity of the disorder. The most important long-term complication of the conditioning regime is the occurrence of cancer, and it is possible that genetic predisposition and components of the conditioning regime contribute to an increased frequency of tumours even though tumours do occur in the absence of radiation.68 It is dicult to see how, with the heterogeneity of FA described above, a single conditioning regime will be suitable for all patients.69 A new cytoreductive regimen has been introduced for transplants using non-sibling donors. The procedure uses T-cell-depleted stem-cell transplants following a ¯udarabine based cytoreduction regimen and this has led to reports of rapid engraftment with minimum toxicity.69,70

BLOOM SYNDROME Bloom syndrome is a very rare disorder although in the Askenazic Jewish population the incidence is much higher. The clinical features, particularly in children, are very typical and include a narrow face with sun-sensitive telangiectatic erythema on the butter¯y area, and small stature. Birth weight is lower than normal, and the small stature also results from a post-natal growth de®ciency. There may be congenital abnormalities present and an immunode®ciency leading to serious infections. Intelligence is normal; fertility is normal in females but adult males appear to be infertile, and Bloom syndrome is associated with a high incidence of cancer. The spectrum of tumour types includes leukaemias, and a range of carcinomas and embryonic tumours with some individuals having more than one primary tumour.71 Chromosomes in Bloom syndrome Lymphocytes from Bloom syndrome show several characteristic chromosomal features. First there is an increase in the level of gaps and breaks, second an increase in chromatid interchanges (particularly quadriradial chromosomes involving both homologues of a pair) and, most striking of all, a very high frequency of spontaneously occurring sister chromatid exchanges (SCE)72±up to ten times more than the SCE rate in normal lymphocytes. In addition to the characteristic clinical features, demonstration of a high level of SCE can provide a rapid con®rmation of the diagnosis. The occurrence of increased numbers of quadriradials and SCE is a consequence of a high recombination rate between sister chromatids and between homologous chromosomes and, although the molecular mechanism underlying the generation of SCEs is still uncertain, study of the Bloom syndrome protein, BLM, will clarify this. Sensitivity to environmental agents Bloom syndrome cells in culture have been reported to be sensitive to UV light as well as to several chemical agents, including mitomycin C, N-nitroso-N-ethylurea and ethyl methane sulphonate, but not ionizing radiation.73,74

Chromosome instability syndromes 639

The BLM gene The gene for Bloom syndrome on chromosome 15q75 has been cloned and is a homologue of the Escherichia coli recQ genes. Returning BLM cDNA to Bloom syndrome cells corrected the high number of SCE and restored BLM protein expression in the nucleus.76,77 BLM is normally located in the nucleus as both discrete nuclear foci and in a di€use distribution. Further work has shown that the nuclear foci associated with BLM are nuclear domain 10 bodies (promyelocytic leukaemia nuclear bodies).78 In addition, BLM may co-localize with some telomere sequences, although there is no evidence of a role for BLM in maintaining telomere length.78 There are four other human genes in the RecQ family: RecQL/RecQ1, WRN, RecQ4 and RecQ5. The genes mutated in Werner's syndrome and Rothmund± Thomson syndrome are WRN79 and RECQ480 respectively, which are also both homologues of the E. coli recQ gene. WRN is located in the nucleolus81, which is di€erent from the major sites for BLM, although BLM does co-localize with WRN in the nucleolus during S phase.78 The RecQ proteins, including BLM82 are all ATPdependent helicases working in a 30 ! 50 direction. In the absence of the RecBC pathway of homologous recombination, in E. coli, the RecF pathway is used as an alternative and RecQ is a component of this pathway.83 The RecQ DNA helicase suppresses illegitimate recombination in E. coli.84 The RecF pathway proteins have speci®c functions in normal cells, including the repair of broken replication forks. Bacterial RecQ, together with RecA and single-stranded DNA binding protein (SSB), is an initiator of homologous recombination and can also disrupt recombination intermediates.83 The free DNA ends at a stalled replication fork may anneal to each other, forming a Holliday junction.85 It is suggested that, normally, BLM recognizes the Holliday junction and catalyses reverse branch migration, thus restoring the replication fork.86 In Bloom syndrome cells without BLM, and the absence of reverse branch migration, the Holliday junctions will be resolved, leading to recombinant DNA molecules.86 BLMÿ/ÿDT40 chicken cells showed an increase in SCE, as might be expected, but in contrast Rad54ÿ/ÿBLMÿ/ÿ double-knockout DT40 cells showed a much reduced frequency of SCEs, suggesting that a large fraction of SCEs in the BLMÿ/ ÿ cells occur through homologous recombination and involvement of Rad54.87 BLM, therefore, appears to act during DNA replication and has an anti-recombinase role. BLM may also have a role in the meiotic recombination process.88 BLM physically interacts with replication protein A89 which stimulates its helicase activity on DNA duplexes.

BLM in haematopoietic cells BLM expression has been examined in human haematopoietic cells. The sequences of the CDR3 region VDJ rearrangement in peripheral B cells of Bloom's patients were in frame, and N region insertions were present, suggesting normal VDJ recombination and, therefore, that BLM is not involved directly in VDJ recombination.90,91 In experiments examining the presence of illegitimate recombination between the TCRg and b chain genes some evidence of abnormal T cell rearrangements of the type also seen in A-T T cells was observed, although not to the same extent.91 BLM does not appear to be involved in immunoglobulin hypermutation. BS V regions displayed the normal distribution of mutations, indicating that the defect in BS is not related to the mechanism of somatic hypermutation.92

640 A. M. R. Taylor

Blm null mouse Viable Blm null mice have been generated recently that are prone to a variety of tumours.93 Cell lines from these mice showed increased rates of mitotic recombination but not meiotic recombination. Gene targeting eciency was higher in the Blm null ES cells compared with wild-type ES cells, consistent with observations that SCE is mediated by homologous recombination. Blm de®ciency also led to an increased rate of somatic loss of heterozygosity (LOH), which was shown to result in an increase in adenomatous polyps in ApcMin mice on a Blm null background.93 LOH is therefore the likely cause of the tumours in the Blm null mice.

SUMMARY The diagnosis of these disorders is now fairly straightforward and, for all of them, there are good con®rmatory laboratory tests for the diagnosis. These still rely on an initial chromosomal analysis, for sensitivity to IR (A-T) or MMC or DEB (FA) or for an increased frequency of SCE (BS), as these can all be completed quickly. Further analysis can be undertaken to show either the total loss or decrease in the level of expression of the appropriate protein in the patient's cells. Finally, mutation screening can identify the particular mutations carried by the patient although, because of the size of the coding region, absence of common mutations and diculties in methodologies, this can take some time with no guarantee of identifying mutations. There is no treatment for A-T, and stem-cell transplantation using HLA-matched unrelated donors for FA remains problematic. Tumour development is a major feature of these disorders. The genes are cloned, however, and this has allowed progress in understanding their normal function. It is only very recently, however, that some understanding has been gained of how the FANC genes relate to each other. An area of interest is the role that some of these genes might have in the wider population±in particular, the role of ATM or FANC genes in sporadic tumours, including leukaemias. At the beginning of the chapter the di€erences between the A-T and A-T-like group of disorders, Fanconi anaemia and Bloom syndrome were stressed and the subsequent details have emphasized these di€erences. However, the question can be asked: are there, nevertheless, some similarities between these disorders, and what conclusions can we draw from consideration of them? The broad similarities are (i) the presence, in each disorder, of characteristic chromosomal abnormalities in peripheral blood lymphocytes, (ii) an unusual cellular sensitivity to an environmental agent, (iii) a predisposition to cancer±again, the spectrum of tumours is di€erent in each of these three groups of disorders, and (iv) in each case mutation in a gene which is involved in DNA repair, replication or recombination. With respect to the genes concerned it is clear that there is evidence from all three disorders for a defect involving homologous recombination repair. Recently a BRCA1associated genome surveillance complex (BASC) was identi®ed which contains at least 15 subunits.94 These included BRCA1, ATM, BLM and the Mre11/Rad50/Nbs1 complex. As noted above, BRCA1 also co-localizes with the FANCD2 protein. In addition, BLM co-localizes with BRCA194 and so does Mre11. Recently it has been reported that the ATM-dependent phosphorylation of BLM occurs in response to ionizing radiation, suggesting that BLM acts as a downstream e€ector of ATM.95 All the genes associated with the chromosome instability syndromes appear to be involved in monitoring damage in S phase at replication forks and initiating repair of

Chromosome instability syndromes 641

abnormal structures at compromised replication forks. This does not preclude an involvement in other forms of repair. Some reports, for example, suggest that lack of a functional FANC gene results in loss of ®delity of DNA end-joining in speci®c DNA double-strand breaks and possibly in VDJ recombination.96 Following on from the defect in recombination repair, the mechanism by which a tumour forms is di€erent in each disorder. In the case of A-T, particular chromosome translocations are important for the development of tumours, while in Bloom syndrome loss of heterozygosity in somatic cells may contribute to tumorigenesis. There are other genes associated with homologous recombination repair, but null mutations of these often result in lethality.97 In the hMRE11/hRAD50/NBS1 complex, hMRE11 and NBS1, while lethal in the homozygous null state in animals, are still present in the human population as hypomorphic mutations.33,39 Mutations in other recombination repair genes may similarly be present in the population. A challenge is to identify the patients carrying them. Acknowledgements I gratefully acknowledge the support of the Cancer Research Campaign, the Kay Kendall Leukaemia Fund, the Ataxia Telangiectasia Society (UK), and the Medical Research Council.

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