Outcomes of mismatched and unrelated donor hematopoietic stem cell transplantation in Fanconi anemia conditioned with chemotherapy only.

Fanconi anemia (FA) is a genomic instability syndrome associated with bone marrow failure, myelodysplastic syndrome (MDS), and/or acute myeloid leukemia (AML) requiring hematopoietic stem cell transplantation (HSCT) to restore normal hematopoiesis. Although low-intensity fludarabine-based preparative regimens without radiation confer excellent outcomes in FA HSCTs with HLA-matched sibling donors, outcomes for FA patients with alternative donors are less encouraging, albeit improving. We present our experience with 17 FA patients who completed mismatched related or unrelated donor HSCT using a non-radiation fludarabine-based preparative regimen at Charité University Medicine Berlin. All patients engrafted; however, one patient had unstable chimerism in the setting of multi-viral infections that necessitated a stem cell boost to revert to full donor chimerism. Forty-seven percent of patients developed grade I acute graft-verus-host disease (aGVHD). No grade II-IV aGVHD or chronic graft-versus-host disease of any severity occurred. At a median follow-up of 30 months, 88 % of patients are alive with normal hematopoiesis. Two patients died of infections 4 months post-transplantation. These results demonstrate that short-term outcomes for FA patients with mismatched and unrelated donor HSCTs can be excellent using chemotherapy only conditioning. Viral reactivation, however, was a major treatment-related complication.

siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors.

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/- female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/- females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/- mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/- versus C57BL/6J-Trp53+/- mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/-, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/- by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/- mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Shared Copy Number Variation in Simultaneous Nephroblastoma and Neuroblastoma due to Fanconi Anemia.

Concurrent emergence of nephroblastoma (Wilms Tumor; WT) and neuroblastoma (NB) is rare and mostly observed in patients with severe subtypes of Fanconi anemia (FA) with or without VACTER-L association (VL). We investigated the hypothesis that early consequences of genomic instability result in shared regions with copy number variation in different precursor cells that originate distinct embryonal tumors. We observed a newborn girl with FA and VL (aplasia of the thumbs, cloacal atresia (urogenital sinus), tethered cord at L3/L4, muscular ventricular septum defect, and horseshoe-kidney with a single ureter) who simultaneously acquired an epithelial-type WT in the left portion of the kidney and a poorly differentiated adrenal NB in infancy. A novel homozygous germline frameshift mutation in PALB2 (c.1676_c1677delAAinsG) leading to protein truncation (pGln526ArgfsX1) inherited from consanguineous parents formed the genetic basis of FA-N. Spontaneous and induced chromosomal instability was detected in the majority of cells analyzed from peripheral lymphocytes, bone marrow, and cultured fibroblasts. Bone marrow cells also showed complex chromosome rearrangements consistent with the myelodysplastic syndrome at 11 months of age. Array-comparative genomic hybridization analyses of both WT and NB showed shared gains or amplifications within the chromosomal regions 11p15.5 and 17q21.31-q25.3, including genes that are reportedly implicated in tumor development such as IGF2, H19, WT2, BIRC5, and HRAS.

Measuring neuropsychological change following breast cancer treatment: an analysis of statistical models.

This article considers the quantitative techniques currently in use in the evaluation of cognitive impairments associated with chemotherapy treatment for breast cancer. To illustrate differences among analytical approaches, all analyses were applied to baseline and posttreatment scores on neuropsychological tests obtained from Stages I and II breast cancer patients receiving either chemotherapy or hormonal therapy; a healthy control group with similar demographics to those of the treatment groups was also included. Conventional group analyses were compared with individual-based analyses (standardized regression-based and reliable change methods). Both univariate and multivariate techniques with and without covariates produced negligible effects. In contrast, results of the individual-based analyses identified a subset of participants in the chemotherapy group who experienced a severe decline in function on two or more tests. Differences between the control and treatment groups were greater than differences between the treatment groups alone. The standardized regression-based approach was more sensitive than the reliable change index in detecting chemotherapy and hormonal therapy subjects whose performance was different from baseline scores on two or more tests (roughly 80% vs. 50% of participants). From a clinical perspective, the degree of impairment determined on the basis of the individual-based methodologies could have a major impact on quality of life for those affected. On the whole, we argue that the standardized regression-based approach, allowing for the assessment of individual practice effects and evaluation of moderator variables, is the method of choice in this context.

Microarray mRNA expression analysis of Fanconi anemia fibroblasts.

Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation.

LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration. In the present work, we analysed the effect of LASP-1 on biology and function of human ovarian cancer cell line SKOV-3 using small interfering RNA technique (siRNA). Transfection with LASP-1-specific siRNA resulted in a reduced protein level of LASP-1 in SKOV-3 cells. The siRNA-treated cells were arrested in G(2)/M phase of the cell cycle and proliferation of the tumour cells was suppressed by 60-90% corresponding to around 70% of the cells being transfected successfully as seen by immunofluorescence. Moreover, transfected tumour cells showed a 40% reduced migration. LASP-1 silencing is accompanied by a reduced binding of the LASP-1-binding partner zyxin to focal contacts without changes in actin stress fibre and microtubule organisation or focal adhesion morphology as observed by immunofluorescence. In contrast, silencing of zyxin is not influencing cell migration and had neither influence on LASP-1 expression nor actin cytoskeleton and focal contact morphology suggesting that LASP-1 is necessary and sufficient for recruiting zyxin to focal contacts. The data provide evidence for an essential role of LASP-1 in tumour cell growth and migration, possibly through influencing zyxin localization.

Fanconi anemia: causes and consequences of genetic instability.

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.

Screening Fanconi anemia lymphoid cell lines of non-A, C, D2, E, F, G subtypes for defects in BRCA2/FANCD1.

Biallelic mutations in BRCA2/FANCD1 were recently recognized as a rare cause of Fanconi anemia (FA). Using immunodetection with an antiserum directed against the carboxyterminus of the BRCA2 protein, we screened 38 lymphoid cell lines from FA patients whom we could not previously assign, via retroviral complementation analysis, to any of six known FA complementation groups (FA-A, -C, -D2, -E, -F, or -G). Three of these 38 cell lines lacked the 380-kDa BRCA2 signal on immunoblots. DNA sequencing showed biallelic compound and truncating mutations in two of the immuno-negative cell lines, whereas a monoallelic frameshift mutation and an amino acid substitution were detected in the third cell line. Our data show that less than 10% of unassigned FA cell lines harbor truncating mutations in BRCA2/FANCD1. This finding strongly suggests the existence of (an) additional, as yet unknown FA gene(s).

Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction.

Fanconi anemia (FA) is a genetically and phenotypically heterogenous autosomal recessive disease associated with chromosomal instability and hypersensitivity to DNA crosslinkers. Prognosis is poor due to progressive bone marrow failure and increased risk of neoplasia, but revertant mosaicism may improve survival. Mechanisms of reversion include back mutation, intragenic crossover, gene conversion and compensating deletions/insertions. We describe the types of reversions found in five mosaic FA patients who are compound heterozygotes for single base mutations in FANCA or FANCC. Intragenic crossover could be shown as the mechanism of self-correction in the FANCC patient. Restoration to wildtype via back mutation or gene conversion of either the paternal or maternal allele was observed in the FANCA patients. The sequence environments of these mutations/reversions were indicative of high mutability, and selective advantage of bone marrow precursor cells carrying a completely restored FANCA allele might explain the surprisingly uniform pattern of these reversions. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensating missense mutation 15 amino acids downstream of the constitutional mutation, which explains the reversion to MMC resistance of the respective lymphoblastoid cell line. With one exception, our mosaic patients showed improvement of their hematological status during a three- to six-year observation period, indicating a proliferative advantage of the reverted cell lineages. In patients with Fanconi anemia, genetic instability due to defective caretaker genes sharply increases the risk of neoplasia, but at the same time increases the chance for revertant mosaicism leading to improved bone marrow function.

Response to X-irradiation of Fanconi anemia homozygous and heterozygous cells assessed by the single-cell gel electrophoresis (comet) assay.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure and cancer susceptibility. Patient cells are sensitive to a variety of clastogens, most prominently cross-linking agents. Although there is the long-standing clinical impression of radiosensitivity, in vitro studies have yielded conflicting results. We exposed peripheral blood mononuclear cells from FA patients and carriers to x-rays and determined their DNA damage and repair profiles using the alkaline single-cell gel electrophoresis (comet) assay. Studies were carried out in two independent series of experiments by two laboratories using different protocols. The cells of both FA patients and carriers showed uniformly high initial DNA damage rates as assessed by the total initial tail moment. In addition, the average residual tail moment at 30 to 50 minutes and the repair half-time parameters were significantly elevated. These findings suggest an increased release of fragmented DNA following x-ray exposure in cells that carry one or two mutations in one of the FA genes. The comet assay may be a useful adjunct for heterozygote detection in families of FA patients.

Sources of variation in patient response to radiation treatment.

PURPOSE: To investigate sources of variation in radiosensitivity displayed by cancer patients and blood donors using the leukocyte apoptosis assay. METHODS AND MATERIALS: Probes were obtained from 105 healthy blood donors, 48 cancer patients displaying normal sensitivity to radiotherapy, 12 cancer patients displaying hypersensitivity to radiotherapy, 12 Ataxia telangiectasia blood donors, and 4 additional individuals with genetic diseases of potentially modified radiosensitivity; 2 neurofibromatosis (NF) donors, a Nijmegen breakage syndrome (NBS) donor, and an Immunodeficiency, Chromosome fragility, Facial anomaly syndrome (ICF) donor. Heparinized blood was diluted in medium, irradiated, and left to incubate for 48 h. CD4 and CD8 T-lymphocyte DNA was stained with propidium iodide and the cells were analyzed by flow cytometry. RESULTS: Radiation-induced apoptosis depended on age and cell type. Cohorts of hypersensitive cancer patients, NBS and AT donors displayed compromised apoptotic response. An asymmetric apoptotic response of T-lymphocytes was observed in an ICF donor and a cryptic hypersensitivity donor. Two NF donors displayed no abnormal sensitivity to radiotherapy but compromised apoptotic T-cell response to X-rays. CONCLUSION: Our studies reveal 4 physiologic sources of variation in radiation response-2 are genetic: cryptic hypersensitivity and hereditary disease, and 2 are epigenetic: cell type and donor age. They emphasize the important role of proteins involved in the recognition and repair of DNA double-strand breaks in determining the response of individuals to radiotherapy.

Functional expression of chimeric receptor genes in human T cells.

Tumor immunotherapy has been limited to date by the poor antigenicity of most tumors, the immunocompromised state of many cancer patients, and the slow tumor penetration and short half-life of exogenously-introduced anti-tumor antibodies. Our group has developed a model immunotherapy system using a chimeric construct containing an antibody V region fused to a T cell activation molecule (T body) introduced by transfection into cytotoxic T cell lines, or populations of activated primary T or natural killer (NK) cells. In this study we have optimized the conditions needed for efficient transduction of human peripheral lymphocytes (PBL) using retroviral vectors pseudotyped with the gibbon ape leukemia virus (GaLV) envelope. Selection of packaging cells producing high virus titers was performed following transfection with constructs containing the green fluorescent protein (GFP), and FACS sorting. As a model chimeric receptor gene we used a tripartite construct consisting of a single-chain anti-TNP antibody variable region linked to part of the extracellular domain and the membrane spanning regions of the CD28 coreceptor molecule and joined at its 5' end to a gene fragment encoding the intracellular moiety of the gamma activation molecule common to the Fcepsilon and Fcgamma receptors. Enriched preparations of retrovectors containing this chimeric receptor and the GFP gene could stably and efficiently transduce human PBL co-activated by anti-CD3 and anti-CD28 antibodies. In routine experiments, the transgene was expressed in 35-70% of the human T cells. Such lymphocytes express the chimeric receptors on their surface and upon stimulation with hapten immobilized on plastic they can produce IL-2. Transfectomas activated in this manner also undergo specific proliferation in the absence of exogenous IL-2. Moreover, the transduced lymphocytes could effectively lyse target cells expressing the TNP hapten on their surface. These studies establish the conditions for the optimal transfection of effector lymphocytes to redirect them against a variety of tumor targets.

WISH-PC2: a unique xenograft model of human prostatic small cell carcinoma.

Prostatic small cell carcinoma is an aggressive subtype of prostate cancer that usually appears as a progression of the original adenocarcinoma. We describe here the WISH-PC2, a novel neuroendocrine xenograft of small cell carcinoma of the prostate. This xenograft was established from a poorly differentiated prostate adenocarcinoma and is serially transplanted in immune-compromised mice where it grows within the prostate, liver, and bone, inducing osteolytic lesions with foci of osteoblastic activity. It secretes to the mouse Chromogranin A and expresses prostate plasma carcinoma tumor antigen-1, six-transmembrane epithelial antigen of the prostate, and members of the Erb-B receptor family. It does not express prostate-specific antigen, prostate stem cell antigen, prostate-specific membrane antigen, and androgen receptor, and it grows independently of androgen. Altogether, WISH-PC2 provides an unlimited source in which to study the involvement of neuroendocrine cells in the progression of prostatic adenocarcinoma and can serve as a novel model for the testing of new therapeutic strategies for prostatic small cell carcinoma.

A heterozygous frameshift mutation in the Fanconi anemia C gene in familial T-ALL and secondary malignancy.

BACKGROUND: Patients with Fanconi Anemia (FANC) have a well documented increased risk to develop malignancies, especially Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). The risk for heterozygous individuals is not clear, epidemiological data are inconsistent. If the risk for heterozygous individuals to develop malignancies was increased, they should be found in groups of patients with AML or MDS at higher proportion than in the normal population. We are currently screening a pediatric population with hematologic malignancies for mutations in the FANCA, FANCC and FANCG gene, and report here on siblings carrying a heterozygous frameshift mutation in the FANCC Gene. PATIENTS AND METHODS: Using PCR based single strand conformational analysis we screened the DNA from pediatric patients suffering from 1 degree or 2 degrees MDS, CMML/JMML or AML for mutations in the FANCA (43 exons), FANCC (14 exons) and FANCG (14 exons) gene, and included one patient with refractory T-ALL, being the brother of a patient with T-ALL and MDS transforming into AML. Aberrant PCR products were directly sequenced. Flowcytometric measurement of mitogen-sensitivity and G2-phase arrest is used to evaluate cultured stimulated lymphocytes from individuals carrying FANC-mutations. RESULTS: A novel heterozygous frame-shift mutation, 377-378delGA in the FANCC gene was found in 2 siblings, both suffering from T-ALL with subsequent MDS transforming to AML in one of them. No other mutation was found by direct sequencing of the complete FANCC gene. Both patients died under therapy. The parents (first degree cousins) and one healthy brother are also carriers. Their lymphocytes show a higher mutagen sensitivity than normal, but do not get blocked in G2 phase as being typical for Fanconi Anemia. CONCLUSION: As the mutation causes a premature Stopcodon within exon 4 of the FANCC gene it has to be regarded as a causal FANCC gene defect. The findings within this family support the hypothesis of an increased risk to develop malignancies in heterozygous carriers of FANC-mutations. A systematic screening of further patients is needed, and we are currently examining a larger cohort to get a better estimate of the true risk of heterozygosity.

Sequence analysis of the ATM gene in 20 patients with RTOG grade 3 or 4 acute and/or late tissue radiation side effects.

PURPOSE: Patients with ataxia-telangiectasia (A-T) show greatly increased radiation sensitivity and cancer predisposition. Family studies imply that the otherwise clinically silent heterozygotes of this autosomal recessive disease run a 3.5 to 3.8 higher risk of developing cancer. In vitro studies suggest moderately increased cellular radiation sensitivity of A-T carriers. They may also show elevated clinical radiosensitivity. We retrospectively examined patients who presented with severe adverse reactions during or after standard radiation treatment for mutations in the gene responsible for A-T, ATM, considering a potential means of future identification of radiosensitive individuals prospectively to adjust dosage schedules. MATERIAL AND METHODS: We selected 20 cancer patients (breast, 11; rectum, 2; ENT, 2; bladder, 1; prostate, 1; anus, 1; astrocytoma, 1; Hodgkins lymphoma, 1) with Grade 3 to 4 (RTOG) acute and/or late tissue radiation side effects by reaction severity. DNA from the peripheral blood of patients was isolated. All 66 exons and adjacent intron regions of the ATM gene were PCR-amplified and examined for mutations by a combination of agarose gel electrophoresis, single-stranded conformational polymorphism (SSCP) analysis, and exon-scanning direct sequencing. RESULTS: Only 2 of the patients revealed altogether four heteroallelic sequence variants. The latter included two single-base deletions in different introns, a single-base change causing an amino acid substitution in an exon, and a large insertion in another intron. Both the single-base deletions and the single-base change represent known polymorphisms. The large insertion was an Alu repeat, shown not to give rise to altered gene product. CONCLUSIONS: Despite high technical efforts, no unequivocal ATM mutation was detected. Nevertheless, extension of similar studies to larger and differently composed cohorts of patients suffering severe adverse effects of radiotherapy, and application of new technologies for mutation detection may be worthwhile to assess the definite prevalence of significant ATM mutations within the group of radiotherapy patients with adverse reactions. To date, it must be recognized that our present results do not suggest that heterozygous ATM mutations are involved in clinically observed radiosensitivity but, rather, invoke different genetic predisposition or so far unknown exogenous factors.

Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules.

Characterization of ATM gene mutations in 66 ataxia telangiectasia families.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.