Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene. (1/692)

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.  (+info)

The Fanconi anemia group E gene, FANCE, maps to chromosome 6p. (2/692)

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disease with bone marrow failure and predisposition to cancer as major features, often accompanied by developmental anomalies. The cells of patients with FA are hypersensitive to DNA cross-linking agents in terms of cell survival and chromosomal breakage. Of the eight complementation groups (FA-A to FA-H) distinguished thus far by cell fusion studies, the genes for three-FANCA, FANCC, and FANCG-have been identified, and the FANCD gene has been localized to chromosome 3p22-26. We report here the use of homozygosity mapping and genetic linkage analysis to map a fifth distinct genetic locus for FA. DNA from three families was assigned to group FA-E by cell fusion and complementation analysis and was then used to localize the FANCE gene to chromosome 6p21-22 in an 18.2-cM region flanked by markers D6S422 and D6S1610. This study shows that data from even a small number of families can be successfully used to map a gene for a genetically heterogeneous disorder.  (+info)

Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. (3/692)

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently cloned FANCG/XRCC9 protein is required for binding of the FANCA and FANCC proteins. Moreover, the FANCG protein is a component of a nuclear protein complex containing FANCA and FANCC. The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex. Our results demonstrate that the three cloned FA proteins cooperate in a large multisubunit complex. Disruption of this complex results in the specific cellular and clinical phenotype common to most FA complementation groups.  (+info)

Loss of FancC function results in decreased hematopoietic stem cell repopulating ability. (4/692)

Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue of FANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC -/- cells in vivo. Competitive repopulation assays were conducted where "test" FancC -/- or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC -/- BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 x 10(5) competitor cells. We determined that FancC -/- cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+ cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function.  (+info)

Expression of the Fanconi anemia group A gene (Fanca) during mouse embryogenesis. (5/692)

About 80% of all cases of Fanconi anemia (FA) can be accounted for by complementation groups A and C. To understand the relationship between these groups, we analyzed the expression pattern of the mouse FA group-A gene (Fanca) during embryogenesis and compared it with the known pattern of the group-C gene (Fancc). Northern analysis of RNA from mouse embryos at embryonic days 7, 11, 15, and 17 showed a predominant 4.5 kb band in all stages. By in situ hybridization, Fanca transcripts were found in the whisker follicles, teeth, brain, retina, kidney, liver, and limbs. There was also stage-specific variation in Fanca expression, particularly within the developing whiskers and the brain. Some tissues known to express Fancc (eg, gut) failed to show Fanca expression. These observations show that (1) Fanca is under both tissue- and stage-specific regulation in several tissues; (2) the expression pattern of Fanca is consistent with the phenotype of the human disease; and (3) Fanca expression is not necessarily coupled to that of Fancc. The presence of distinct tissue targets for FA genes suggests that some of the variability in the clinical phenotype can be attributed to the complementation group assignment.  (+info)

Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts. (6/692)

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.  (+info)

Cyclophosphamide metabolism in children with Fanconi's anaemia. (7/692)

Although patients with Fanconi's anaemia (FA) exhibit a heightened sensitivity to DNA cross-linking agents, modified doses of CY continue to be used in their conditioning prior to BMT. We measured the pharmacokinetics and metabolism of CY in six children with FA using an established high performance thin layer chromatography technique. CY doses ranged between 5 and 20 mg/kg (median 10 mg/kg). The median CY clearance was 0.6 l/h/m2 (range 0.4-1.1 l/h/m2), t1/2 was 8.1 h (range 6.7-9.5 h) and volume of distribution was 0.19 l/kg (range 0.16-0.34 l/kg), respectively. These results contrast with those previously reported from a comparable group of non-FA children in whom the median CY clearance was 3.2 l/h/m2 (range 2-5 l/h/m2) (P = 0.035), t1/2 was 2.4 h (range 2-3.8 h) (P = 0.035) and volume of distribution 0.5 l/kg (range 0.26-0.95 l/kg) (NS). Unlike the control group in whom the presence of inactive metabolites of CY was common, metabolites could not be found in any FA patient. The enhanced sensitivity of children with FA to CY may in part result from altered drug metabolism.  (+info)

Delayed engraftment and mixed chimerism after HLA-identical sibling donor BMT in Fanconi anaemia. (8/692)

A 12-year-old girl with Fanconi anaemia (FA) received a bone marrow transplant from her HLA-identical brother following conditioning with cyclophosphamide (20 mg/kg), thoraco-abdominal radiation (TAI) (4 Gy) and equine anti-thymocyte globulin (ATG) (90 mg/kg). Engraftment was delayed and initially tenuous, and was followed by mixed chimerism (MC) over a follow-up period of 2 years. DNA analysis of engraftment was performed on whole peripheral blood and on separated granulocytes, B and T lymphocytes using PCR detection of CA tandem repeat polymorphisms. At 10 weeks post BMT, granulocytes were predominantly donor, but B and T lymphocytes recipient, in origin. Over the subsequent 90 weeks, granulocytes and B lymphocytes were donor-derived, whilst T cells showed persistent MC but with an increasing donor component. Marrow haemopoietic function (Hb, ANC and platelet count) improved gradually in parallel with a rise in the proportion of donor lymphocyte engraftment. We postulate that a population of recipient lymphocytes survived conditioning and in turn delayed the development of full donor chimerism. Although transient MC has been described after allogeneic BMT in FA, its association with delayed engraftment, and persistence for more than 1 year post BMT, has not been documented clearly.  (+info)