Bose, M., Smith, L.E., Petrini, J., Concannon, P., and W.F. Morgan
Genomic instability, profound radiosensitivity and cancer predisposition characterize Nijmegen Breakage Syndrome (NBS). The protein product deficient in NBS (Nbs1) is an important component of the Mre11/Rad50/ Nbs1complex. This complex is involved in the recognition, signaling and repair/processing of damage induced by ionizing radiation. Patients having NBS have two homozygous mutant copies of the nbs1 gene and therefore make no functional p95 protein. The most common mutation in nbs1 is a 5 bp deletion in exon 6 resulting in a frameshift mutation. While the disease NBS is rare, it is estimated that a significant proportion of the population may be heterozygous at this locus and represent a potentially radiation sensitive "normal" population. Humans will always be exposed to radiation, and the goal of the DOE is to ensure that human health is adequately and appropriately protected. NBS heterozygous individuals may be particularly susceptible to environmental, diagnostic or therapeutic exposures involving ionizing radiation; furthermore, heterozygous individuals may experience an increased risk from ionizing radiation at doses that are below current workplace exposure limits.
The purpose of this research funded by the DOE is to improve the scientific basis for understanding potential risks to a sensitive population from low dose radiation exposure using a mouse model for heterozygosity at the nbs1 gene. Drs. Petrini and Concannon have developed mice that are homozygous mutant and heterozygous at the nbs1 gene. The mutation in the Petrini mice mimics a mutation observed in patients with NBS. Consistent with the existence of patients having a deletion in exon 6 in both copies of the nbs gene, homozygosity of the mutation in this mouse strain is not embryonically lethal. Additionally, we have obtained a heterozygous mouse strain from Dr. Concannon which has a deletion that includes 1 Kb of upstream promoter sequences and exon 1 of the nbs gene. The deletion of promoter sequences and exon 1 is embryonically lethal. Both knockout (KO) strains were created through gene targeting and the genotype of any mouse can be easily determined by PCR amplification.
One of the most important aspects in identifying a radiation sensitive portion of the human population is to have a rapid, reliable and relatively non-invasive assay for heterozygosity at nbs1. Currently, the only way to identify heterozygous individuals for any genetic disease is to know the parents of an affected child. Nbs1 is relatively small gene spanning approximately 50 Kb of genomic DNA and encoding a 2.4 kb transcript representing 16 exons, however, sequencing nbs1 to determine heterozygosity is too expensive and time consuming to be feasible as a screening method.
A functional assay for nbs1protein may be a more appropriate screening method for heterozygosity. In unirradiated wildtype (WT) cells, immunofluoresence (using antibodies against the proteins Mre11, Rad50, and Nbs1) demonstrates a diffuse staining within the nucleus. Eight hours after exposure to ionizing radiation, the Mre11/Rad50/Nbs1 complex is found in discrete foci. This pattern presumably represents recruitment of the complex to DNA damage for repair. Foci response is dependent on the genetic background of the cells examined and NBS cells do not form foci after irradiation. In preliminary experiments using IMR90 human fibroblasts, formation of foci exhibits a linear dose response. We will examine foci response in our heterozygous and homozygous NBS mice to determine whether there are quantitative or qualitative differences relative to WT controls. Preliminary attempts to examine foci formation in mouse blood have been unsuccessful and indeed lymphoblastoid cells in general appear refractory to foci formation. To this end we are isolating fibroblasts from mouse-tail snips and growing the cells in chambered slides for analysis of foci.
Because NBS is characterized by chromosomal instability, we will monitor instability in heterozygous mice after exposure to low doses of radiation using a micronucleus assay (see figure 1). In preliminary experiments, WT mice were exposed to 0, 1, 10 and 100cGy whole body irradiation. A minimum of ten mice were used per dose. Peripheral blood samples were taken from mouse tail bleeds at one month and 3 months post-irradiation. The blood was smeared onto acridine orange coated slides and total number of micronuclei were counted per 1000 polychromatic erythrocytes. These data indicate that there is a linear dose response at both 1 month and 3 months post-irradiation (see figure 2). Additionally, no significant decrease in the number of micronuclei was observed at 3 months after irradiation.
The goal of these studies is to accurately identify heterozygotes and to evaluate whether radiation-induced genomic instability in heterozygous animals can be correlated with increased cancer risk. One unirradiated nbs heterozygous mouse has developed testicular cancer. The tumor was removed and will be examined by a pathologist. We are currently breeding the heterozygous mice and expect to begin proposed experiments when we have at least 30 confirmed heterozygous mice from each strain.
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| Figure 1. Micronuclei in mouse polychromatic erythrocytes (arrowed).
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Figure 2. Histogram of micronuclei frequency. 1 month (red) and 3 months (blue) post-irradiation |