Carol Reifsteck

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.

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links.

ABSTRACT Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G2/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G1, S, and G2 cells were treated with photoactivated 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G2 did not result in a G2/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G1 did not cause a G1/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to ∼2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex.

OBJECTIVE Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. MATERIALS AND METHODS We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. RESULTS Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. CONCLUSIONS These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.

Identification, genomic organization and mRNA expression of CRELD1, the founding member of a unique family of matricellular proteins.

We have isolated and characterized a unique gene that encodes a highly conserved membrane bound extracellular protein that defines a new epidermal growth factor-related gene family. The CRELD1 (Cysteine-Rich with EGF-Like Domains 1) gene (previously known as cirrin) was cloned from a human chromosome 3 BAC. Mapping of the gene confirmed its position at chromosome 3p25.3. The gene is ubiquitously expressed in early development and later becomes more markedly expressed in the developing heart, limb buds, mandible and central nervous system. Expression persists in adulthood in most tissues. Sequence analysis suggests that this is a cell adhesion protein. The mouse orthologue was cloned and mapped to the syntenic region of mouse chromosome 6. Orthologues or homologues have also been identified for cow, Chinese hamster, Drosophila and Caenorhabditis elegans. The CRELD1 gene is deleted in the human cytogenetic disorder 3p- syndrome and is in the region of loss of heterozygosity for several types of cancer. A potential role for this protein in these disorders is discussed.

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3.

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.

Transgene Insertion Induced Dominant Male Sterility and Rescue of Male Fertility Using Round Spermatid Injection

Abstract Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFRα2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.

Immortalization of four new fanconi anemia fibroblast cell lines by an improved procedure

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hapered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D.

COMPLEMENTATION GROUP ASSIGNMENTS IN FANCONI ANEMIA FIBROBLAST CELL LINES FROM NORTH AMERICA

Fanconi anemia is a rare autosomal recessive disease characterized by developmental defects of the thumb and radius, childhood onset of pancytopenic anemia and increased risk of leukemia. At least five complementation groups (A-E) have been defined but only theFAC gene has been cloned. Cells can be assigned to complementation group C by direct mutation analysis. To facilitate the search for additional FA genes and to measure the frequency of complementation groups, we have established new genetically marked immortalized FA-A and FA-D fibroblast cell lines and show their usefulness as universal fusion donors. These reference FA cell lines facilitated somatic cell fusion analysis and enabeled us to assign the complementation group in 16 unrelated FA patients from North America. The majority of patients, belong to FA complementation group A (69%), followed by FA-C (18%), FA-D (4%) and FA-B or FA-E (9%).

FANCONI ANEMIA GROUP A AND D CELL LINES RESPOND NORMALLY TO INHIBITORS OF CELL CYCLE REGULATION

Cells from patients with Fanconi anemia (FA) show decreased viability and decreased chromosome stability after treatment with DNA cross-linking agents, compared to normal cells. FA cells also show a relative accumulation at the G2/M transition after such treatment. This has suggested a possible checkpoint abnormality. In the studies presented here, treatment with hydroxyurea, caffeine or inhibitors of cell cycle kinases did not reveal abnormalities in survival or chromosome stability in FA-A or FA-D cells. Chromosomal breaks introduced by hydrogen peroxide or methyl methanesulfonate accumulated to the same extent in FA-A or FA-D cells as in normal cells. We conclude that FA-A and FA-D cells respond normally to agents known to alter the cell cycle or introduce DNA strand breaks. FA cells process strand breaks and a variety of DNA monoadducts normally. Our results are compatible with repair of DNA crosslinks being slower in FA than in normal cells and FA cells having normal cell cycle checkpoints.

Molecular pathogenesis of secondary myeloid leukemia: Resistance to inhibitory cytokines and autocrine vegf-dependence of the UOC-M1 cell line

Abstract Monosomy 7 (−7) is common in alkylating agent induced- or Fanconi anemia-related myelodysplastic syndrome and acute myeloid leukemia. Interested in using genetic complementation to discover tumor suppressor genes on chromosome 7 that may play a role in leukemogenesis, we are characterizing the phenotype of a factor-independent, megakaryoblastoid, CD34+ cell line (UoC-M1) with complex cytogenetics including (−7). UoC-M1 cells were resistant to 100ng/ml IFNγ and 5ng/ml TGFβ in colony forming unit assays. In contrast, TNFα inhibited growth and resulted in caspase 3 activation. UoC-M1 cells produced highly vascularized, solid tumors in NOD/SCID mice. The tumor cell karyotype was identical to UoC-M1. Supernatants from unstimulated UoC-M1 cells in vitro demonstrated VEGF production (240pg/ml/72 hours from 1×10 5 cells by ELISA) but no GM-CSF, FLT-3, SCF, IL-6, TPO, TNFα, or IL-1β. Normal human CD34+ cells yielded 4pg/ml VEGF/10 6 and 57pg/ml/10 6 when stimulated with 200U/ml rhGM-CSF for 3 days. UoC-M1 also expresses both VEGFR1 (FLT-1) and VEGFR2 (KDR). Because VEGF normally varies inversely with von Hippel-Lindau protein (VHL), we analyzed UoC-M1 mRNA for VHL and the VHL-interacting factors CUL2, HIF1α/β, elongin B/C, and SP1. All were constitutively expressed. We conclude that TGF and IFNγ resistance, VHL resistant-VEGF production, autocrine growth, and in vivo tumorigenesis will be of potential value in genetic complementation studies for the identification of tumor suppressor genes on chromosome 7.