Ataxia telangiectasia (AT) patients have inactivating mutations in both copies of the ATM gene. The ATM protein that the gene encodes is involved in DNA double-strand break (DSB) recognition; in its absence, p53 response to DSBs is delayed and reduced. In addition,AT patients have a high propensity for cancer, and cells from these patients show chromosomal instability. Here, using an in vivo mouse model system with the pink-eyed unstable mutation,we demonstrate that the absence of functional Atmresults in a significantly elevated frequency of intrachromosomal recombination resulting in deletion events (wild-type 17.73%,heterozygous Atm 15.72%, and mutant Atm30.33%). No such increase was observed in mice heterozygous for Atm. These results further advocate the role of ATM in maintaining genomic integrity after the onset of endogenous damage. This system relies on the initiation of events during a relatively short time frame to produce an observable deletion product. AT patients have a lifelong exposure to endogenous damage and perhaps similarly acting external agents. Because 25% of our genome consists of repeated elements, genomic instability due to an increased level of homologous recombination between such repeats, as observed here, may contribute to carcinogenesis in AT patients.

AT3is an autosomal recessive pleiotropic human disease. Phenotypic manifestations beyond cerebellar degeneration and telangiectasia(dilated blood vessels) include thymic degeneration, immune deficiency,retarded growth, premature aging, gonadal dysgenesis, an acute sensitivity to ionizing radiation and chromosomal instability (for reviews see Refs. 1, 2). In addition, patients display an extreme predisposition to lymphoreticular malignancies (3).

The gene mutated in AT patients was recently identified by positional cloning (4). This has allowed molecular analyses of the gene and its protein product. More recently, the mouse Atmgene was located and deleted to produce knock-out mice (5, 6, 7, 8). The mouse models display many of the pleiotropic phenotypes of the patients and seem to be a good model system of the disease.

ATM is a member of the phosphatidylinositol 3-kinase family (4). This is an extensive family of proteins with either lipid or protein kinase activity. The family member most similar to ATM is the catalytic-subunit of the DNA-PKcs. DNA-PKcs is involved in the processing of DSBs, in particular those produced during V(D)J recombination (for review see Ref. 9) ATM also appears to have a role in DSB recognition and response. After ionizing radiation and radiomimetic damage, the most relevant biological damage is thought to be DSBs. How this damage is recognized and repaired is still under investigation. It is clear, however, that the major damage recognition pathway that responds to DSBs involves p53 phosphorylation that results in the stabilization and activation of p53 (10, 11). p53 then initiates either an apoptotic response or cell cycle arrest presumably to allow DNA repair reactions to be effected. In cells from AT patients, p53 stabilization and activation are both delayed and reduced, suggesting a role for ATM earlier in the same pathway (12, 13, 14).

Cells from AT patients display chromosomal instability both spontaneously and after induction by ionizing radiation or radiomimetic agents (reviewed in Refs. (15, 16). Cytogenetic analysis revealed a higher spontaneous incidence of chromosome breaks,chromosome gaps, acentric fragments, dicentric chromosomes, and aneuploidy. In addition, T lymphocytes have an elevated frequency of translocations with break points mapping to the T-cell antigen receptor genes and the immunoglobulin heavy chain genes (reviewed in Ref. 16). After exposure to ionizing radiation or radiomimetic agents, cells from AT patients have an increased frequency of chromosomal aberrations compared with normal cells. This susceptibility to DSB-inducing agents has led to numerous detailed in vitrostudies.

In the present study, we examined the effects of Atm status on in vivo recombination frequencies in the mouse. We chose a mouse model that facilitates detection of recombination events between an intrachromosomal tandem duplication. The mouse mutation pun results from a direct tandem duplication of 70 kb within the p gene (Fig. 1,A; Refs. 17, 18). p encodes a melanosomal integral membrane protein. In the absence of a functional p gene, mice have pink eyes and a dilute coat color. The pun mutation spontaneously reverts to wild-type by deletion of one copy of the 70-kb repeat (Fig. 1). A reversion event within a melanocyte results in ptranscription and, thus, in a pigmented cell. When a deletion/reversion event occurs in a premelanocyte of the mouse embryo, the melanocyte has the potential to continue proliferating and to differentiate into a clone of revertant melanocytes. The resultant fur-spots have been previously confirmed by molecular analysis as being due to pun reversion (19, 20, 21).

The pun mutation has previously been used to determine frequencies of recombination in the mouse (21, 22, 23). Approximately 5–10% of pun mice in a pure C57BL6/J genetic background spontaneously contain fur-spots (17, 21). This frequency of reversion is increased by treatment with various carcinogenic agents (21, 22, 23).

In the present work, we determine the effect of the loss of functional Atm on the spontaneous frequency of DNA deletions at the pun locus in vivo. We have bred mice homozygous for the punmutation and heterozygous for the Atm mutation. These mice have been crossed with each other to produce Atm wild-type,heterozygous, or mutant littermates. The frequency of mice that demonstrate pun reversion events has been determined for each genotype.

Mouse Strains.

NIH Black Swiss Atm +/− (5) mice were crossed into the C57BL/6J pun/pun(22) genetic background by three backcrosses. The resulting pun/pun, Atm +/− mice had a genetic background containing about 87.5% of C57BL/6J, and were morphologically similar to the parental C57BL/6J pun/pun. The pun/pungenotype was observed phenotypically in the progeny of the second backcross as mice with a dilute (gray) coat color.

PCR Genotype.

The Atm genotype was determined by PCR amplification as described previously (24) with the exception that PCR cycling was performed for 35 cycles at 94°C for 30 s and 62°C for 2 min. DNA was prepared from tail biopsies by standard protocols. PCR amplification was performed in a Perkin-Elmer 9700 Thermocycler. Three PCR reactions were performed for each DNA sample, one with wild-type specific primers, another with mutant specific primers, and a final reaction with both wild-type and mutant primers together.

pun Reversion Assay.

A pun reversion event in the premelanocyte of an embryo results in a patch of black fur on the offspring. When 10 days old, the size and position of any fur spot was noted, their presence was then confirmed at weaning. The protocol used in this test is similar to the “mouse spot test” (for review see Refs. 25, 26). Matings were set up between pun/punAtm+/− mice resulting in littermates homozygous for pun and all of the three Atm genotypes. The combined area of the spots on a mouse is denoted as “spot area.”

Statistical Analysis.

Comparison between numbers of events was done by a Gtest (27). The G test is equivalent to a contingency chi-square test but allows for classes with zero events.

Fewer Than Expected Atm Mutant Offspring.

C57BL/6J pun/punAtm heterozygous mice were bred to obtain progeny that were wild type, heterozygous, or mutant for Atm. Surprisingly, when the Atm genotype was determined for each of the progeny,29.4% were wild type, 54.3% heterozygous, and 16.3% mutant for Atm (Table 1). This is significantly different from the expected Mendelian segregation pattern of 25% wild-type, 50% heterozygous, and 25%mutant Atm mice (G, 17.9; P ≤ 0.005), with the difference being due to the number of observed Atm mutant mice. A reduced Atm mutant-mouse frequency has not been reported with outbred strains (5, 6), which suggests a difference with inbred strains or the C57BL6/J background in particular.

Atm Mutants Have an Increased Frequency of Intrachromosomal Recombination.

Each 10-days-old mouse was examined for the presence of fur spots, the indication of a pun reversion event. The frequency of mice with reversion events of each genotype is presented in Table 1. Wild-type and Atm heterozygous mice show no difference in spotting frequency. In contrast, mice mutant for Atm have a significantly increased spotting frequency compared with both the wild-type (P < 0.01)and the Atm heterozygous mice (P < 0.005). These data strongly suggest that the lack of functional ATM renders a cell susceptible to spontaneous genomic instability, DNA deletion events in particular.

The frequency of pun reversion was also determined by gender within each genotype (Table 2). No significant difference was observed in the spotting frequency between genders of the same genotype. Similarly, there was no significant difference in the spotting frequency of wild-type and Atm heterozygous mice of the same gender. As before,however, the spotting frequency between Atm mutant mice and either wild-type or Atm heterozygous mice was significantly elevated for both genders. The results demonstrated that there was no gender bias in the distribution of spots.

To determine whether there was any bias in the frequency of events within the population due to a clustering of events in the offspring of particular mice, a more detailed examination of the data was undertaken. Litters were examined individually but displayed no obvious clustering of reversion frequencies. In addition, all of the litters derived from an individual male or female were examined. Again, the results did not indicate any apparent founder effect throughout the population.

Increased Frequency of Late Reversion Events in Atm−/− Mice.

To examine further the timing of the recombination events that led to pun reversions, the total area of dark fur spots on each mouse (henceforth, “spot area”) was determined. Because premelanocytes expand in a clonal fashion during embryogenesis,the spot area is thought to reflect the relative time of the initial reversion deletion event. As the population of premelanocytes increases over time, the larger the spot area and the earlier in embryogenesis a single deletion event is likely to have occurred. The average spot areas of the spotted wild-type, heterozygous, and mutant Atmmice are 52.3, 29.4, and 33.8 mm2, respectively. To examine these differences further, spotted mice were grouped by the size of spot area and by genotype. The results of this analysis are presented in Table 3. Mice were grouped as having spot areas either larger or smaller than three arbitrarily chosen areas, 10, 50, and 75 mm2 in size. This division revealed some significant differences in the spot area between genotypes. In particular, the proportion of small spot area (<75 mm2) to large spot area (≥75 mm2) was significantly increased in Atm heterozygous or mutant mice compared with wild-type mice(Ratio of smaller:larger being 11.80 and 17.50, respectively, compared with 3.33). This result indicates that the increased frequency of spotted mice in the Atm mutant mice may be due to an increased frequency of smaller spot areas.

The difference in the frequency of spot areas of a particular size was examined further. The frequency of mice with small spot areas (<10,<50, and <75 mm2) was determined as a proportion of all of the mice of a particular genotype (spotted and not spotted); the results are shown in Table 4. In summary, there is a highly significant difference between Atm mutant mice and either the wild-type or the Atm heterozygous mice for the three groupings. A similar analysis with the frequency of large spot areas (≥10, ≥50,and ≥ 75 mm2) as a proportion of all of the mice of a particular genotype (spotted and not spotted) was examined as well. The frequency of larger spot areas was not significantly different between the Atm mutant mice and the other two genotypes for either the 50-mm2 or the 75-mm2 size groupings (not shown). The apparent intermediate effect with the Atm heterozygous mice (see Table 3) seems to be due to a lower frequency of large spot areas rather than to the increased frequency of smaller spot areas as observed for the mutant Atm mice.

Atm mutant mice display a retarded growth (5, 6), and this reduced body size might account for the disproportionate increase in Atm mutant mice with smaller spot areas as opposed to all of the sizes of spot area. The difference in body size of an Atm mutant mouse has been reported to be 0.8–0.9 times the size of heterozygous and wild-type littermates (5, 6). Measurements from this laboratory revealed similar growth retardation. Taking the spot areas measured for the mutant mice and multiplying by 1.25 should compensate for such retarded growth. After this calculation, the frequency of mice with spot areas smaller than 10 mm2 among the total number of mice of a particular genotype is no longer significantly different between either the wild-type or the Atm heterozygous mice and the Atm mutant mice (G, 1.87 and 1.22, respectively). There is, however, still a highly significant increase between these pairs when examining spot areas less than 50 mm2and 75 mm2 [wild-type/mutant G, 8.80(P, <0.005) and G, 10.08 (P < 0.005), respectively; heterozygous/mutant G, 9.69(P, <0.005) and G, 10.67 (P < 0.005), respectively]. Therefore, the retarded growth of the Atm mutant mouse cannot account for the difference in reversion area observed. Interestingly the frequency of mice with spot areas ≥10 mm2 is now significantly different between wild-type or Atm heterozygous and Atmmutant mice. This suggests that the average size of the increased number of spot events is approximately 10 mm2.

In summary, these results suggest that Atm mutant mice have a frequency of large spot areas similar to that of wild-type mice, but have a significantly increased frequency of smaller spot areas arising from an increased number of reversion events at a particular stage of embryogenesis.

We found a significantly lower than expected frequency of Atm mutant offspring (about 16% versus the expected 25%) from crossing Atm heterozygous mice. A reduced Atm mutant mouse frequency has not been reported with outbred strains (5, 6). Our mice consisted of about 87.5% C57BL/6J background. For segregation of the Trp53allele, others (28, 29) reported a loss of Trp53 mutant offspring in inbred backgrounds with a gender bias, predominantly linked to female-associated defects in neural tube closure. In a similar manner, this was not found for an outbred background (30). Examining the gender of our Atm mutant offspring did not reveal any gender bias (Table 2).

The results from this study clearly demonstrate that there is an increased frequency of pun reversion events in mice lacking ATM (Table 1). Two possible explanations for the observed increase in pun reversion events may be an increased level of initiating DNA damage or that there is a change in DSB repair response in AT cells. In addition, the size distribution of the spot areas observed on Atm mutant mice was disproportionately smaller than on wild-type and Atmheterozygous mice (Table 4). These results are discussed below.

Deletion events between duplicated DNA sequences have been extensively studied in a number of different model systems, called the yeast DEL assays and the human cell culture DEL assay, involve recombination substrates similar to the pun fur-spot assay, a gene interrupted by a duplication of internal sequences (31, 32). Spontaneous deletion by homologous recombination between the duplicated regions reverts the interrupted gene. The frequency of such deletions can be induced to higher levels by treatment with various mutagenic agents, including radiomimetic agents and alkylating agents (31, 33, 34). Most interestingly, oxidative mutagens are potent inducers of DEL recombination in yeast (35, 36, 37).

Previously, we have examined the frequency of pun reversion events after treatment with carcinogens (21, 22, 23). In these and more recent studies, we observed an increased number of reversion events with an approximate spot area of 10 mm2 after genotoxic carcinogen exposure on 10 days post-coitum.4We found that the average size of the additional events in the ATM mutant background is also about 10 mm2. This suggests that the increased number of reversion events observed in Atm mutant mice in the current study is the result of initial reversion events that occur either 10 dpc or later during gestation.

Deletion events occur spontaneously, probably as the result of endogenous DNA damage. One source of DNA damage is oxidative stress, a byproduct of normal cellular processes that include respiration,steroidogenesis, β-oxidation of high molecular weight fatty acids,polyamine oxidation, and iron metabolism. The reactive oxygen species produced can chemically attack DNA leading to base modifications,oxidation of nucleotides, single-strand breaks, and double-strand breaks (for a review see Ref. 38). As a consequence,constant surveillance and repair must maintain genomic integrity within the cell.

It has been postulated that cells from AT patients have a higher than normal basal level of oxidative stress (39). In addition it has been postulated that the age-dependant increase of oxidizing proteins and mutations in DNA is related to a lifetime of exposure to oxidative stress (40). The premature aging observed with AT patients may result from an accelerated cellular degeneration from higher than normal exposure to oxidative stress. Furthermore,ATM-deficient cells are more sensitive to agents that produce oxidative damage but respond normally when exposed to other agents that can directly modify DNA. Rotman and Shiloh (39) discuss several studies in light of the uncoupling of ATM as an early sensor of reactive oxygen species and/or oxidative damage and normal response pathways. For instance, NF-κB plays a key role in regulating gene expression in response to oxidative stress. In the absence of ATM,NF-κB is constitutively activated indicating a constant state of oxidative stress (41).

The spot size that was increased in Atm mutant mice was the same as the spot size that was induced by carcinogens at the 10 dpc. It is, therefore, likely that the high frequency of reversion events in Atm mutant mice may be initiated at about 10 dpc. Interestingly, embryos are more susceptible to prooxidant exposure on 12 dpc compared with maternal tissues (42). Before this susceptibility, an increase in glutathione peroxidase and superoxide dismutase activity is observed (43), possibly to counter increasing levels of endogenous oxidative damage. Also, by 10.5 dpc, Atm mRNA is detectable, ubiquitously so by 13.5 dpc (44). If ATM is normally involved in counteracting an increased level of endogenous oxidative damage at this time, its deficiency may result in higher than normal levels of DNA damage.

An alternative, but not mutually exclusive explanation to the increased frequency of recombination events, may be that the response to DSBs is deficient or abnormal in ATM cells compared with normal cells. Analysis of the kinetics of DSB repair has revealed that AT cells have defective fast repair whereas the slow component is normal (45). Meyn (46) reported a 30- to 200-fold increase in the spontaneous frequency of homologous intrachromosomal deletion events at an introduced substrate in human cells. Others have reported that the frequency of linear plasmid repair is decreased or is not altered, but the fidelity of nonhomologous recombination repair is compromised (47, 48, 49, 50, 51). If ATM-deficient cells are less able to perform illegitimate end-joining, then a DNA substrate that can be repaired by either the homologous or illegitimate end-joining pathway in a normal cell may favor repair by the homologous pathway in an ATM-deficient cell. Because the punassay system detects homologous deletion recombination events, any abnormal preference for homologous recombination over illegitimate recombination would appear as an increased frequency of pun reversions. Thus, there need not be an increase in DNA damage, but merely a difference in the processing to produce an increased frequency of pun reversions. This possibility,however, does not explain the disproportionate increase of spots smaller than 10 mm2 in the Atm-mutant mice. Thus, a difference in processing recombination substrates may act in concert with an increased level of endogenous DNA damage at about 10 days post coitum.

From the results presented in this study, it can be concluded that homologous intrachromosomal recombination events leading to pun reversions occur at an elevated frequency in mice mutant for Atm compared with mice that are wild-type or heterozygous. Considering that about 25% of the human genome consists of repetitive DNA sequences in either tandem repeats or interspersed repetitive elements (52), there are plenty of potential substrates for intrachromosomal homologous recombination events (for example, see Ref. 53). In contrast, a recently completed study using the Aprt mutation assay system demonstrated no increased mutation frequency in Atm mutant mice compared with wild-type mice (54). Together these results suggest that homologous intrachromosomal recombination events leading to genome rearrangements may be more important for carcinogenesis in AT patients than mutations.

Fig. 1.

The pun mutation and reversion to wild-type. A, a schematic representation of the pun mutation. The mutation is the result of a spontaneous duplication of 70 kb within the gene duplicating exons and introns 6–18. B, a schematic representation of the revertant/wild-type p gene. Reversion of pun to p must be by deletion between the homologous internal repeat regions to produce a functional wild-type gene. The possible mechanisms of such recombination events have been investigated previously (55, 56).

Fig. 1.

The pun mutation and reversion to wild-type. A, a schematic representation of the pun mutation. The mutation is the result of a spontaneous duplication of 70 kb within the gene duplicating exons and introns 6–18. B, a schematic representation of the revertant/wild-type p gene. Reversion of pun to p must be by deletion between the homologous internal repeat regions to produce a functional wild-type gene. The possible mechanisms of such recombination events have been investigated previously (55, 56).

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1

Supported by American Cancer Society Grant RPG-95-076-04-MGO, National Institute of Environmental Health Sciences,NIH, RO1 Grant ES09519 and KO2 award ES00299 (to R. H. S), and National Institutes of Health Research Cancer Development Award F32GM19147 (to A. J. R. B.).

3

The abbreviations used are: AT, ataxia telangiectasia; ATM/Atm, human/mouse ataxia telangiectasia mutated; DSB, double-stranded DNA break; DNA-PKcs, DNA-dependent protein kinase catalytic-subunit; pun,pink-eyed unstable; SSB, single-stranded DNA break; DEL,deletion.

4

A. J. R. Bishop and R. H. Schiestl, unpublished observations.

Table 1

Comparison of pun reversion frequency in mice wild-type, heterozygous, and mutant for Atm

Mice homozygous for pun and heterozygous for Atm were crossed with each other and their progeny were examined. The number of spotted and nonspotted mice was determined and grouped according to genotype for comparison (see lines).

GenotypeWild typeHeterozygousMutant
No. of mice examined 220 407 122 
Spotted mice, n (%) 39 (17.73) 64 (15.72) 37 (30.33) 
Nonspotted mice, n 181 343 85 
    
    
Significance between pairs    
G test, P ≤ (GNSa <0.01 <0.005 
 (0.41) (7.01) (11.97) 
GenotypeWild typeHeterozygousMutant
No. of mice examined 220 407 122 
Spotted mice, n (%) 39 (17.73) 64 (15.72) 37 (30.33) 
Nonspotted mice, n 181 343 85 
    
    
Significance between pairs    
G test, P ≤ (GNSa <0.01 <0.005 
 (0.41) (7.01) (11.97) 
a

NS, not significant.

Table 2

Comparison of pun reversion frequency by genotype and gender

The number of spotted and nonspotted mice was determined and grouped according to gender and genotype.

GenotypeWild TypeHeterozygousMutant
No. of male mice examined 98 216 67 
Spotted mice, n (%) 14 (14.29) 31 (14.35) 18 (26.87) 
Nonspotted mice, n 84 185 49 
    
    
Significance between indicated pairs    
G test, P ≤ (GNSa <0.05 <0.025 
 (0.00) (3.96) (5.18) 
No. of female mice examined 122 191 55 
Spotted mice, n (%) 25 (20.49) 33 (17.28) 19 (34.55) 
Nonspotted mice, n 97 158 36 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.05 <0.01 
 (0.51) (3.87) (7.04) 
GenotypeWild TypeHeterozygousMutant
No. of male mice examined 98 216 67 
Spotted mice, n (%) 14 (14.29) 31 (14.35) 18 (26.87) 
Nonspotted mice, n 84 185 49 
    
    
Significance between indicated pairs    
G test, P ≤ (GNSa <0.05 <0.025 
 (0.00) (3.96) (5.18) 
No. of female mice examined 122 191 55 
Spotted mice, n (%) 25 (20.49) 33 (17.28) 19 (34.55) 
Nonspotted mice, n 97 158 36 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.05 <0.01 
 (0.51) (3.87) (7.04) 
a

NS, not significant.

Table 3

Comparing area of reversion in spotted mice wild type, heterozygous, or mutant for Atm

The total “spot area” was determined for each spotted mouse; the resulting values were grouped according to size and genotype of mouse. Genotypes were then compared.

GenotypeWild TypeHeterozygousMutant
Total spotted mice 39 64 37 
No. of spot areas <10 mm 17 36 19 
No. of spot areas ≥10 mm 22 28 18 
Ratio of smalllarge 0.77 1.29 1.06 
    
    
    
Significance between indicated pairs    
G test, P ≤ (GNSa NS NS 
 (1.56) (0.46) (0.23) 
No. of spot areas <50 mm 29 56 34 
No. of spot areas ≥ 50 mm 10 
Ratio of smalllarge 2.90 7.00 11.33 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.05 NS 
 (2.82) (4.32) (0.48) 
No. of spot areas <75 mm 30 59 35 
No. of spot areas ≥75 mm 
Ratio of smalllarge 3.33 11.80 17.50 
    
    
Significance between indicated pairs    
G test, P ≤ (G<0.05 <0.025 NS 
 (4.65) (5.15) (0.22) 
GenotypeWild TypeHeterozygousMutant
Total spotted mice 39 64 37 
No. of spot areas <10 mm 17 36 19 
No. of spot areas ≥10 mm 22 28 18 
Ratio of smalllarge 0.77 1.29 1.06 
    
    
    
Significance between indicated pairs    
G test, P ≤ (GNSa NS NS 
 (1.56) (0.46) (0.23) 
No. of spot areas <50 mm 29 56 34 
No. of spot areas ≥ 50 mm 10 
Ratio of smalllarge 2.90 7.00 11.33 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.05 NS 
 (2.82) (4.32) (0.48) 
No. of spot areas <75 mm 30 59 35 
No. of spot areas ≥75 mm 
Ratio of smalllarge 3.33 11.80 17.50 
    
    
Significance between indicated pairs    
G test, P ≤ (G<0.05 <0.025 NS 
 (4.65) (5.15) (0.22) 
a

NS, not significant.

Table 4

Comparing area of reversion smaller than 10, 50, or 75 mm2 in all mice wild type, heterozygous, or mutant for Atm

The total “spot area” was determined for each spotted mouse; the resulting values were grouped according to size and genotype of mouse. The frequency of each spot area category per total number of mice is shown.

GenotypeWild TypeHeterozygousMutant
Total No. of mice 220 407 122 
No. of spot areas <10 mm 17 36 19 
Frequency 7.73% 8.85% 15.57% 
    
    
Significance between indicated pairs    
G test, P ≤ (GNSa <0.05 <0.05 
 (0.23) (4.90) (4.20) 
No. of spot areas <50 mm 29 56 34 
Frequency 13.18% 13.76% 27.87% 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.005 <0.005 
 (0.04) (10.86) (12.11) 
No. of spot areas <75 mm 30 59 35 
Frequency 13.64% 14.50% 28.69% 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.005 <0.005 
 (0.09) (11.14) (11.88) 
GenotypeWild TypeHeterozygousMutant
Total No. of mice 220 407 122 
No. of spot areas <10 mm 17 36 19 
Frequency 7.73% 8.85% 15.57% 
    
    
Significance between indicated pairs    
G test, P ≤ (GNSa <0.05 <0.05 
 (0.23) (4.90) (4.20) 
No. of spot areas <50 mm 29 56 34 
Frequency 13.18% 13.76% 27.87% 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.005 <0.005 
 (0.04) (10.86) (12.11) 
No. of spot areas <75 mm 30 59 35 
Frequency 13.64% 14.50% 28.69% 
    
    
Significance between indicated pairs    
G test, P ≤ (GNS <0.005 <0.005 
 (0.09) (11.14) (11.88) 
a

NS, not significant.

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