The Fanconi anemia gene product FANCF is a flexible adaptor protein

The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.     

Positional cloning of a novel Fanconi anemia gene, FANCD2

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.     

A comparison of topoisomerase I activity in normal and transformed cells

Many viral oncogenes encode protein~yrosine kinase activities. However, importantin vivo substrates of these enzymes have yet to be identified. Recently, type I topoisomerases were shown to bein vitro substrates for two tyrosine kinases. Following tyrosine phosphorylation, topoisomerase I activity was reduced 10-fold (Tse-Dinhet al. Nature 312:785–786, 1984). To determine whether topoisomerase I activity was modulated by tyrosine phosphorylationin vivo, we have measured topoisomerase I activity in nuclear lysates prepared from both normal fibroblasts and cells transformed by two different viral oncogenes (v-abl, v-src). Under a variety of experimental conditions, we have found no evidence to support the notion that type I topoisomerase activity is modulated by tyrosine phosphorylationin vivo.     

Studies of gene transfer and reversion to mitomycin C resistance in Fanconi anemia cells

As a first step to the cloning of the Fanconi anemia (FA) gene, we have attempted to correct the sensitivity of FA cells to DNA crosslinking agents by the introduction of wild-type DNA. The protocol involved the introduction of both genomic and pRSVneo DNA, selection for G418-resistant colonies and the subsequent selection of mitomycin C-resistant cells from the latter. Preliminary experiments indicated that untransformed FA cells were not suitable recipients for the introduction of foreign DNA, so all experiments were performed with an SV40-transformed FA cell line. Approximately 40,000 G418-resistant colonies were obtained in 5 separate experiments at an overall frequency of about 5 X 10(-4). These were then selected in mitomycin C and 15 colonies were recovered. Colonies were obtained with wild-type DNA (both human and rodent) and with FA DNA at about the same frequency of 2 X 10(-7). Colonies were isolated and shown to have a stable, partial (from 25 to 90% of wild-type) resistance to mitomycin C. One colony was also shown to be partially resistant to two other DNA crosslinking agents, diepoxybutane and nitrogen mustard. This clone also had an intermediate level of spontaneous and MMC-induced chromosome aberrations. pRSVneo, but not rodent, DNA could be demonstrated in the high molecular weight fraction of several colonies. Thus, it is likely that these colonies represent partial revertants rather than transfectants. These mitomycin C-resistant FA cells should be useful for the biochemical analysis of the FA mutation.     

Mutation analysis of the Fanconi anemia gene FACC.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by a unique hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C (FACC) has recently been cloned. We have amplified FACC exons with their flanking intron sequences from genomic DNA from 174 racially and ethnically diverse families in the International Fanconi Anemia Registry and have screened for mutations by using SSCP analysis. We identified eight different variants in 32 families; three were detected in exon 1, one in exon 4, one in intron 4, two in exon 6, and one in exon 14. Two of the eight variants, in seven families, did not segregate with the disease allele in multiplex families, suggesting that these variants represented benign polymorphisms. Disease-associated mutations in FACC were detected in a total of 25 (14.4%) of 174 families screened. The most frequent mutations were IVS4 + 4 A-->T (intron 4; 12 families) and 322delG (exon 1; 9 families). Other, less common mutations include Q13X in exon 1, R185X and D195V in exon 6, and L554P in exon 14. The polymorphisms were S26F in exon 1 and G139E in exon 4. All patients in our study with 322delG, Q13X, R185X, and D195V are of northern or eastern European or southern Italian ancestry, and 18 of 19 have a mild form of the disease, while the 2 patients with L554P, both from the same family, have a severe phenotype. All 19 patients with IVS4 + 4 A-->T have Jewish ancestry and have a severe phenotype.     

Isolation of a cDNA representing the Fanconi anemia complementation group E gene

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome with at least seven different complementation groups. Four FA genes (FANCA, FANCC, FANCF, and FANCG) have been identified, and two other FA genes (FANCD and FANCE) have been mapped. Here we report the identification, by complementation cloning, of the gene mutated in FA complementation group E (FANCE). FANCE has 10 exons and encodes a novel 536-amino acid protein with two potential nuclear localization signals.     

Screening strategies for a highly polymorphic gene: DHPLC analysis of the Fanconi anemia group A gene

Introduction: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)). Methods: PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice: native, and after adding a WT-PCR product. Retention patterns were compared with previously identified polymorphic PCR products or mutants. Results and discussion: We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Growth of cultured cells from patients with Fanconi anemia

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 ± 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 ± 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM, while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.     

Further studies on compartmentalisation of DNA-topoisomerase I in Fanconi anemia tissue

Summary Previously we found increased DNA-topoisomerase I activity in the cytoplasmic fraction of a mature placenta from a child homozygously affected with Fanconi anemia (FA). Now determination of this enzyme was extended to one heterozygous and four homozygous FA fibroblast cultures in confluent stage, and one mature and one immature placenta from homozygously affected children. In all these cases enzyme activity was found in the cytoplasmic fraction in the range between 0.2 and 1.3 U/g protein. For comparison six normal fibroblast cultures, five normal mature and two immature placentae were studied. The cytoplasmic fractions from all but one fibroblast culture, which showed trace activity, showed no detectable activity. The normal mature placentae had no measurable cytoplasmic activity, while the immature trisomy 18 placentae contained 0.1 and 0.27 U/g protein. In a homozygous FA fibroblast culture the total cellular distribution was determined; the cytoplasmic fraction contained 6.5% of the whole cellular activity.     

Damage-resistant DNA synthesis in Fanconi anemia cells treated with a DNA cross-linking agent

Fanconi anemia (FA) is a recessive disorder associated with diverse congenital anomalies, progressive bone marrow failure, and a marked predisposition to develop cancer. At the cellular level, FA is characterized by a prolonged G(2) phase in proliferating cells and a marked hypersensitivity to both the cytotoxic and the clastogenic effects of agents which produce DNA interstrand cross-links. Treatment with these agents leads to even further prolongation of the G(2) phase in FA cells. We now show that FA cells, from four different complementation groups, fail to decrease their rates of replicative DNA synthesis, as do normal cells, following treatment with a DNA cross-linking agent. This may be responsible for the prolongation of the G2 phase seen in these cells, and suggests that the fundamental defect in response of FA cells to DNA cross-linking agents may be in the S phase, rather than the G(2) phase, of the cell cycle.     

Arrest of S-phase progression is impaired in Fanconi anemia cells

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.     

Growth of cultured cells from patients with Fanconi anemia.

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 +/- 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 +/- 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM; while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.     

The Fanconi anemia gene network is conserved from zebrafish to human

Fanconi anemia (FA) is a complex disease involving nine identified and two unidentified loci that define a network essential for maintaining genomic stability. To test the hypothesis that the FA network is conserved in vertebrate genomes, we cloned and sequenced zebrafish (Danio rerio) cDNAs and/or genomic BAC clones orthologous to all nine cloned FA genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, and FANCL), and identified orthologs in the genome database for the pufferfish Tetraodon nigroviridis. Genomic organization of exons and introns was nearly identical between zebrafish and human for all genes examined. Hydrophobicity plots revealed conservation of FA protein structure. Evolutionarily conserved regions identified functionally important domains, since many amino acid residues mutated in human disease alleles or shown to be critical in targeted mutagenesis studies are identical in zebrafish and human. Comparative genomic analysis demonstrated conserved syntenies for all FA genes. We conclude that the FA gene network has remained intact since the last common ancestor of zebrafish and human lineages. The application of powerful genetic, cellular, and embryological methodologies make zebrafish a useful model for discovering FA gene functions, identifying new genes in the network, and identifying therapeutic compounds.     

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.     

Irreversible repression of DNA synthesis in Fanconi anemia cells is alleviated by the product of a novel cyclin-related gene.

Primary fibroblasts from patients with the genetic disease Fanconi anemia, which are hypersensitive to cross-linking agents, were used to screen a cDNA library for sequences involved in their abnormal cellular response to a cross-linking challenge. By using library partition and microinjection of in vitro-transcribed RNA, a cDNA clone, pSPHAR (S-phase response), which is able to correct the permanent repression of semiconservative DNA synthesis rates characteristic of these cells, was isolated. Wild-type SPHAR mRNA is expressed in all fibroblasts so far analyzed, including those of Fanconi anemia patients. Correction of the abnormal response in these cells appears therefore to be due to overexpression after cDNA transfer rather than to genetic complementation. The cDNA contains an open reading frame coding for a polypeptide of 7.5 kDa. Rabbit antiserum directed against a SPHAR peptide detects a protein of 7.9 kDa in Western blots (immunoblots) of whole-cell extracts from proliferating, but not resting, fibroblasts. The deduced amino acid sequence of SPHAR contains a motif found in the cyclins, and it is proposed that SPHAR acts within the injected cell by interfering with the cyclin-controlled maintenance of S phase. In agreement with this proposal, normal cells transfected with an antisense SPHAR expression vector have a significantly reduced rate of DNA synthesis during S phase and a prolonged G2 phase, reflecting the need for postreplicative DNA processing before entry into mitosis.