Complementation Analysis in Fanconi Anemia: Assignment of the Reference FA-H Patient to Group A

Joenje, Hans; Levitus, Marieke; Waisfisz, Quinten; D’Andrea, Alan D.; García-Higuera, Irene; Pearson, Tommy; Berkel, Carola G.M. van; Rooimans, Martin A.; Morgan, Neil V.; Mathew, Christopher G.; Arwert, Fré

doi:10.1086/303067

Cited by 112 publications

(57 citation statements)

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“…Eight complementation groups have been defined by somatic cell fusion studies (FA-A through FA-G, including FA-D1 and FA-D2) with each complementation group corresponding to a distinct gene. 25,37 The majority of all patients (60%-65%) fall within group FA-A. Seven of the FA genes have been cloned.…”

Section: Genetic Analysis Of a Lymphoblastoid Cell Line From The Samementioning

confidence: 99%

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia

Lensch¹

2003

Blood

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Myelodysplastic and leukemic stem cell clones that evolve in children and adults with Fanconi anemia universally bear complex cytogenetic abnormalities. The abnormalities are generally recurring deletions or chromosomal loss and involve precisely the same chromosomes with the same frequency as has been described in marrow cells from patients with secondary acute leukemia induced by alkylating agents. Reasoning that acquired Fanconi anemia protein dysfunction might contribute to cytogenetic instability in secondary acute myelogenous leukemia (AML) cells, we analyzed leukemic cells bearing characteristic complex cytogenetic defects obtained from a 68-year-old man whose lymphoblasts showed no evidence of Fanconi anemia. Unlike the lymphoblasts, this myeloid leukemia cell line (UoC-M1) was hypersensitive to mitomycin-C (MMC) and diepoxybutane (DEB) and exhibited a marked decrease in nuclear FANCA, FANCG, and FANCD2-L. Retroviral transduction of FANCA significantly reduced MMC sensitivity but FANCF, FANCG, and FANCC did not. Overexpression of FANCA restored levels of both FANCA and FANCG, whereas overexpression of FANCG or IntroductionA variety of molecular defects have been discovered in patients with myeloproliferative syndromes and acute myelogenous leukemia, many of which result in the activation of autonomous signal transduction pathways for growth and survival. [1][2][3][4][5] Although oncogenic mutations in such pathways are sufficient to induce myeloproliferative disorders in transgenic mice, 2,3 the development of acute leukemia requires additional mutations that ultimately interfere with myeloid differentiation. 6 The risk of this second type of mutation would be necessarily enhanced by cytogenetic instability, a manifestation of which in the leukemic clone would be multiple cytogenetic defects. That such complex cytogenetic defects universally occur in the myeloid leukemic clones of children with Fanconi anemia, a cytogenetic instability syndrome, supports this notion. In fact, because the clonal abnormalities found in this clinical context exactly recapitulate those found in patients with secondary myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML), 7,8 and high-risk MDS, 9 we hypothesized that acquired Fanconi anemia (FA) protein dysfunction might serve as a progression factor for the evolution of cytogenetically unstable AML clones, even in patients without hereditary evidence of Fanconi anemia. To test this notion, we examined a variety of AML cells and cell lines, including a cell line from a 68-year-old patient with cytogenetic defects characteristic of secondary AML. The patient's lymphoblasts were normal, but the leukemic cell population exhibited hypersensitivity to mitomycin C, a feature of Fanconi anemia. Nuclear FANCC, FANCG, and FANCA levels were markedly reduced, and retroviral complementation indicated that the leukemic cells exhibited a secondary defect of FANCA function. Because lymphoblasts from this same patient showed normal levels of nuclear Fanconi proteins, we propo...

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Section: Genetic Analysis Of a Lymphoblastoid Cell Line From The Samementioning

confidence: 99%

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia

Lensch¹

2003

Blood

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“…To date at least 11 complementation groups have been defined (5)(6)(7). Eight genes have been cloned (8 -17).…”

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Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

Qiao¹,

Mi²,

Wilson

et al. 2004

Journal of Biological Chemistry

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Fanconi anemia (FA) 1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1-4). To date at least 11 complementation groups have been defined (5-7). Eight genes have been cloned (8 -17). However, the gene products resemble no known proteins and have few identifiable functional protein motifs. One exception is the recently cloned FANCL gene, which contains the ubiquitin ligase motif. In addition, the FANCD1 gene has been identified as BRCA2, one of the familial breast cancer genes.Cells derived from patients with the FA exhibit characteristic hypersensitivity caused by DNA cross-linking agents and generalized decreased survival (18 -22). However, no defined biochemical mechanism for this hypersensitivity has been elucidated, although studies have implicated cytokine dysregulation, excessive, oxidative damage, defects in DNA repair, and lack of cell cycle control (23-27). Patient and cellular phenotypes across all the complementation groups are similar, suggesting an inter-relatedness or cooperativity between the FA proteins.This cooperativity has been borne out by work we have done in showing binding of FANCA and FANCC in a protein complex in both nucleus and cytoplasm (28 -30). Recent work has found the FANCE, FANCF, FANCG, and FANCL proteins in the complex as well (31-34). A large complex is suggested by our recent work (35), and binding does not occur in any of the complementation groups except the FA-D1, D2, I, and J groups (7).One clue to FA function lies in the study of the FANCA protein, which contains a classic bipartite nuclear localization signal and is phosphorylated. Generally, FANCA nuclear localization, phosphorylation, and binding to FANCC are abolished in all complementation groups except the FA-D1 and D2 groups (28 -30, 33). This suggests that a nuclear event is critical to the normal function of the FA proteins, and the aberrant proteins in the FA-D groups may have a role downstream of the FA complex in the nucleus. However, some have found FANCA point mutants that are expressed, translocated to the nucleus, and are phosphorylated to some extent. Some of these mutants are of intermediate MMC sensitivity (36).Over the years, little information has been found that addresses the regulation of the FA proteins. mRNA or protein levels change little in response to DNA damage or the cell cycle. Our recent work has revealed that at least a subset of the FA proteins resides in the nucleus bound to chromatin, where increased protein binding occurs in response to DNA damage (37). One of the non-core complex FA proteins, FANCD2, becomes monoubiquitinated in response to DNA damage (38).In addition, we have shown during the cell cycle that the FA proteins detach from chromatin during mitosis, and FANCG becomes phosphorylated while remaining part of the complex (37). One group has demonstrated that FANCG has a isoform seen in asynchronous cells that is phosphatase sensitive (39).In this paper we report the identification of a phosphopeptide from ...

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“…Seven FA complementation groups (FA-A through FA-G) have been reported (2), with FA-A (Online Mendelian Inheritance in Man, OMIM no. 227650) constituting approximately two-thirds of the patients.…”

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Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Gregory

Wagner

Verlander

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

207

137

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Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815-2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte-macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein-Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colonyforming unit granulocyte-macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA. F anconi anemia (FA) is an autosomal recessive disorder characterized by congenital abnormalities, bone marrow failure, and a predisposition to malignancies, including myelodysplastic syndrome and acute myelogenous leukemia (1). Seven FA complementation groups (FA-A through FA-G) have been reported (2), with FA-A (Online Mendelian Inheritance in Man, OMIM no. 227650) constituting approximately two-thirds of the patients. The FANCA, FANCC, FANCE, FANCF, and FANCG genes have been cloned and mapped to chromosomes 16q24. 3, 9q22.3, 6p21.2-21.3, 11p15, and 9p13, respectively (3-8). However, the specific function of these genes remains unclear.FA cells are uniquely hypersensitive to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin C (MMC) (9). This cellular phenotype can be demonstrated in cultured T cells, B cells, fibroblasts, and fetal cells cultured from amniotic fluid or chorionic villi (10). Hypersensitivity of phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) to DEB is used routinely as a diagnostic test for the syndrome (11). At a DEB concentration that has a minimal effect on normal PBLs (0.1 g͞ml), 80-100% of PBLs analyzed will have chromatid breaks and exchanges in the typical FA patient. However, in a small grou...

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Complementation Analysis in Fanconi Anemia: Assignment of the Reference FA-H Patient to Group A

Cited by 112 publications

References 19 publications

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia

Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

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