Evidence for at least eight Fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.     

Repair analysis of mitomycin C-induced DNA crosslinking in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia patients

The repair of mitomycin C (MMC)-induced DNA crosslinking was analyzed by denaturation-renaturation gel electrophoresis in ribosomal RNA genes in lymphoblastoid cell lines from 4 patients with Fanconi's anemia (FA). In comparison to normal lymphoblastoid cell lines, 2 lines of FA cells belonging to complementation group A clearly exhibited higher sensitivity to MMC and an almost identical deficiency in the removal of DNA crosslinking. A complementation group B cell line, HSC 62, exhibited a lower sensitivity than group A cells and a lesser deficiency in crosslink repair. Another 'non-A' group cell line, HSC 230, reproducibly exhibited even higher sensitivity to MMC than group A cells. The results on MMC crosslinkage removal at the molecular level correlated well with cell survival. The observed subtle differences of repair among the 4 FA cell lines might represent possible genetic differences in the respective FA complementation groups.     

A gene involved in control of human cellular senescence on human chromosome 1q.

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras(+)-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C. This chromosome 1 caused no change in proliferative potential of cell lines representing the other complementation groups. A derivative of human chromosome 1 that had lost most of the q arm by spontaneous deletion was unable to induce senescence in any of the immortal cell lines. This finding indicates that the q arm of human chromosome 1 carries a gene or set of genes which is altered in the cell lines assigned to complementation group C and is involved in the control of cellular senescence.     

Assignment of SV40-Immortalized Cells to More Than One Complementation Group for Immortalization

Human cell lines have been assigned to four complementation groups for immortalization [O.M. Pereira-Smith and J. R. Smith, Proc. Natl. Acad. Sci. USA 85, 6042-6046, 1988]. Three SV40-immortalized epithelial cell lines were fused to cell lines representative of each of these four complementation groups. All three formed senescent hybrids with an SV40-immortalized cell line representative of group A, indicating that SV40 genes do not always cause immortalization via the same genetic mechanism. In contrast to the results of studies with other human cell lines, each of these three cell lines was assigned to more than one complementation group for immortalization. Thus these cell lines may have lost the function of two or more putative senescence genes.     

Molecular genetic approaches to the study of cellular senescence.

Normal cells in culture exhibit limited division potential, which is used as a model for cellular aging. In contrast, tumor-derived, carcinogen- or virus-transformed cells are capable of dividing indefinitely (immortal). Fusion of normal with immortal human cells yielded hybrids having limited life span, indicating that cellular senescence is a dominant phenotype and that immortality is recessive. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. In order to identify the chromosomes and genes involved in growth regulation, that had been modified in immortal cells, we used the technique of microcell fusion to introduce either a normal human chromosome 11 or 4 into cell lines representative of the different complementation groups. Chromosome 11 had no effect on the in vitro life span of the different immortal human tumor lines. However, when a normal human chromosome 4 was introduced into cell lines assigned to complementation group B, the cells lost the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. These results suggest that a gene(s) on human chromosome 4 has been modified in immortal cell lines assigned to complementation group B, to allow escape from senescence. They also provide evidence for a genetic basis for cellular aging.     

The Fanconi anemia group E gene, FANCE, maps to chromosome 6p

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.     

Follow-up analysis of translocation and dicentric frequencies, measured by FISH-chromosome painting in breast cancer patients after partial-body radiotherapy with little bone marrow exposure

Fanconi anemia (FA) is one of several genetic diseases with characteristic cellular hypersensitivity to DNA crosslinking agents which suggest that FA proteins may function as part of DNA repair processes. At the clinical level, FA is characterized by bone marrow failure that affects children at an early age. The clinical phenotype is heterogeneous and includes various congenital malformations as well as cancer predisposition. FA patients are distributed into eight complementation groups suggesting a complex molecular pathway. Three of the eight possible FA genes have been cloned, although their function(s) have not been identified. FA cells are highly sensitive to DNA crosslinking agents (mitomycin C (MMC) and diepoxybutane), with some variability between cell lines. Sensitivity to monofunctional alkylating agents has been reported in some cases, although these studies were performed with genetically unclassified FA cells. To further analyse and characterize the newly identified FA complementation groups, we tested their sensitivity to UV radiation, monofunctional and bifunctional alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA complementation groups D to H show increased sensitivity to the X-ray mimetic drug bleomycin. Furthermore, the single known FA-H cell line shows increased sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in addition to the characteristic sensitivity to crosslinking agents, suggesting a broader spectrum of drug sensitivities in FA cells.     

Genetic heterogeneity of Fanconi's anemia demonstrated by somatic cell hybrids

Summary Cells of patients with Fanconi's anemia (FA) are characterized by their high mitomycin C sensitivity. This specific response was used to study the question of heterogeneity in cell hybrids. After fusion of somatic cells of different FA patients and a normal control, the resulting hybrids were cytogenetically analyzed with respect to their mitomycin C susceptibility. Complementation—indicating heterogeneity—should lead to normal amounts of mitomycin C-induced chromosomal damage. No complementation was found in hybrids between cells of a classical FA patient and one without skeletal malformations. However, clear evidence for heterogeneity was observed in hybrids between cells of the latter patient with early onset and another with late onset of the disease. This confirms the assumption of Schroeder and coworkers based on the high intrafamilial correlation for age at onset.     

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.     

Retroviral gene transfer for the assignment of Fanconi anemia (FA) patients to a FA complementation group

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure, cancer susceptibility, and a variety of developmental defects. The disease is clinically heterogeneous; eight different complementation groups (FA A–H) and, thus, genetic loci have been discovered. Two genes, FAA and FAC, have been cloned. Disease-associated mutations have been detected and rapid mutation screening makes possible the assignment of patients without resorting to time-consuming cell fusion and complementation analysis. Amplification of specific cDNAs from RNA followed by direct or indirect sequence analysis is a standard method for mutation detection. During the course of such examinations of the FAC gene, we have noted that frequently only one of the expressed alleles is successfully amplified. This can lead to false assignment of patients to a complementation group. As we report here, such cases can be rapidly clarified by retroviral gene transfer and complementation analysis. Received: 30 July 1997 / Accepted: 13 October 1997     

Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.     

The Chinese hamster cell mutant V-H4 is homologous to Fanconi anemia (complementation group A)

V-H4, a mitomycin C (MMC)-sensitive Chinese hamster cell mutant, is phenotypically very similar to Fanconi anemia (FA) cells. Genetic complementation analysis shows that V-H4 belongs to the same complementation group as FA group A cells. Proliferating hybrid cell lines obtained after fusion of V-H4 with normal or FA group B cells show an increased resistance to MMC. Absence of complementation was noted in V-H4 x FA group A hybrid cell lines. This was shown not to be due to the absence of a specific human chromosome. The V-H4 mutant represents the first rodent mutant that is genotypically similar to FA complementation group A cells.     

Protoplast fusion — a tool for genetic manipulation and breeding in industrial microorganisms

Protoplasts can be isolated from microbial cells by enzymatic digestion of the cell wall, in the presence of an osmotic stabilizer. Such protoplasts can be induced to fuse in the presence of agents such as the poly (ethylene glycols). When suitably selected auxotrophic strains are used, the fusion products can be recovered by selection on the basis of nutritional complementation. Cultivation of the protoplasts on a hypertonic growth medium induces regeneration of new cell wall material and their subsequent reversion to the normal cell form of the organism. The protoplast fusion technique has been applied sucessfully to both bacterial and fungal systems leading to the recovery of recombinant progeny. In the fungi, the recovery of non-parental segregants from inter-species crosses has also been demonstrated. In assessing the value of the fusion technique, caution may be necessary at this stage in its application to genetic mapping in bacteria. The behaviour of protoplasts, especially with respect to reversion, could be an additional factor that operates during selection, distorting recombination frequencies. However, the fusion technique, in providing a mechanism by which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.     

Mapping of the genes of some components of the electron transport chain (complex I) on the X chromosome of mammals

This paper describes genetic mapping studies with several respiration-deficient mutants of Chinese hamster fibroblasts which have a defect in complex I of the electron transport chain (NADH-coenzyme Q reductase). The mutations associated with two different complementation groups map on the X chromosome. In two cases (G14 and G20) karyotypic and isozyme analyses in hybrids have shown that a gene(s) on the mouse X chromosome complements the mutation(s) in the hamster cell mutant(s). A cosegregation analysis in hybrid cells has shown the corresponding genes to be linked to the HPRT genes (hamster-mouse hybrids of G14, and hamster-hamster hybrids for G14 and G20). By the same method the defective gene in a third mutant (G4) was also shown to be X-linked. A mutation representing a third complementation group (G11) was shown to be on an autosomal gene. These results provide an explanation for our observation that cells with recessive mutations in complementation groups I and II can be selected at relatively high frequencies.     

Fanconi's anemia: what have we learned from the genes so far?

Fanconi's anemia (FA) is a rare genetic disorder affecting children at an early age; patients suffer from progressive bone marrow failure and, in many cases, from congenital malformations. As cells from FA patients have an increased sensitivity to DNA-crosslinking agents, FA has been included among the group of DNA repair disorders. However, identification of a specific DNA repair defect in FA has not been firmly established. None the less, this cellular phenotype has allowed the classification of FA patients into eight complementation groups defining eight possible FA genes. Two of these genes have now been cloned and, although they have raised more questions than they have answered, are facilitating the identification of cellular processes implicated in the pathophysiology of FA, and the design of new therapies.     

Respiration-deficient Chinese hamster cell mutants: genetic characterization.

We present here genetic experiments with a series of Chinese hamster cell mutants defective in oxidative energy metabolism. The mutations were all shown to be recessive in intraspecies hybrids. Thirty-five mutants were sorted into eight complementation groups, but one of these mutants failed to complement representatives of two distinct complementation groups. The possibility was raised that this is a cell carrying two mutations or a deletion. Because of the greatly different frequencies with which such mutants could be isolated from two different Chinese hamster cell lines, CCL16 (DON) and V79, the stability of representatives from each cell line was examined, and it was found that revertants could be obtained after treatment with mutagens, while spontaneous revertants appeared at unmeasurable or extremely low frequencies, with one exception. The mutant with a very noticeable frequency of spontaneous reversion was defective in mitochondrial protein synthesis, and the question arose whether the mutation was on the mitochondrial genome. A detailed fluctuation analysis of reversion rate and comparison with rates for other mutations was consistent with a nuclear mutation. This conclusion was supported by experiments involving fusions with cytoplasts.     

Requirement for ERCC-1 and ERCC-3 gene products in DNA excision repair in vitro. Complementation using rodent and human cell extracts.

Numerous rodent cell lines exist that have defects in nucleotide excision repair of DNA caused by alterations in genes that fall into 10 different complementation groups. The precise roles in the repair of these genes are unknown. We report here that extracts from Chinese hamster ovary cells of excision repair-defective complementation groups 1 and 3 are defective in DNA excision repair in a cell-free system. In vitro complementation can be achieved by mixing extracts from the two groups with one another. In addition, extracts from a human cell line representing xeroderma pigmentosum complementation group B could complement rodent complementation group 1 extracts, but not group 3 extracts. This is consistent with an identity of the ERCC-3 and xeroderma pigmentosum group B genes. Cellular evidence points toward a defect in the incision of damaged DNA in group 1 and 3 mutants. Since the ERCC-1 and ERCC-3 proteins are required for the in vitro reaction, it appears that both gene products are directly involved in the enzymatic incision of damaged DNA, or in preincision reactions. The experiments reported here provide the biochemical basis of an approach to analyze the function of these nucleotide excision repair proteins.     

Fanconi anaemia in Italy: High prevalence of complementation group A in two geographic clusters

Cell fusion studies using lymphoblastoid cell lines from Fanconi anaemia (FA) patients have identified five complementation groups (FA-A to FA-E) among European FA patients. In Italy, of the 45 FA families referred to the Italian Registry of Fanconi Anaemia (RIAF), 15 took part in a project for the identification of complementation groups. Since three immortalized lymphoblast lines were resistant to a cross-linking agent, we analysed only 12 patients by complementation analysis and found that 11 belong to complementation group A. Four and seven families came from two geographic clusters in the Veneto and Campania regions, respectively, which are thought to consist of aggregates of related families in reproductive isolation. The clinical characteristics of the patients showed both intra-and interfamilial heterogeneity, although overall the disease had a relatively mild course. Since the populations in both Veneto and Campania are likely to represent genetic isolates, our finding predicts linkage disequilibrium for markers flanking theFAA gene. DNAs from these FA families may thus be utilized for positional cloning of this gene through haplotype disequilibrium mapping.     

Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.     

Subtyping analysis of Fanconi anemia by immunoblotting and retroviral gene transfer.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Two of the FA genes (FAA and FAC) have been cloned, and mutations in these genes account for approximately 80% of FA patients. Subtyping of FA patients is an important first step toward identifying candidates for FA gene therapy. In the current study, we analyzed a reference group of 26 FA patients of known subtype. Most of the patients (18/26) were confirmed as either type A or type C by immunoblot analysis with anti-FAA and anti-FAC antisera. In order to resolve the subtype of the remaining patients, we generated retroviral constructs expressing FAA and FAC for transduction of FA cell lines (pMMP-FAA and pMMP-FAC). The pMMP-FAA construct specifically complemented the abnormal phenotype of cell lines from FA-A patients, while pMMP-FAC complemented FA-C cells. In summary, the combination of immunoblot analysis and retroviral-mediated phenotypic correction of FA cells allows a rapid method of FA subtyping.