Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes.

Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human-mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins.

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia.

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 FANCC did not restore FANCA levels. The molecular mass of cytoplasmic FANCA, FANCG, FANCC, and nuclear FANCD2 were normal. All exons of FANCA and FANCG were sequenced, and no mutations were found. We conclude that perturbations of as yet unidentified factors that govern the binding activity or intracellular localization of FANCA may promote cytogenetic instability and clonal progression in patients with AML who do not have Fanconi anemia.

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.

Transgene insertion induced dominant male sterility and rescue of male fertility using round spermatid injection.

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, GFR alpha 2 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.