Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.     

Splicing mutations in TP53 in human squamous cell carcinoma lines influence immunohistochemical detection.

The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.     

Point mutation of the p53 gene resulting in splicing inhibition in small cell lung carcinoma

The p53 gene is functionally inactivated mostly by point mutations resulting in amino acid substitutions in a wide variety of human cancers. We found a novel mutation of the p53 gene in a small cell lung carcinoma cell line, Lu-143. One of the allelic p53 genes was lost accompanied by loss of heterozygosity for chromosome 17. In the remaining allelic p53 gene, there was a single-base substitution of G to T at position 1 within the splice donor site of intron 7, and the mutated intron was not spliced out during the mRNA maturation process. As a result of this mutation, larger sized p53 mRNA was expressed and no p53 specific protein was detected in this cell line. These results suggest that mutations causing splicing abnormalities are one of the molecular mechanisms for the p53 gene inactivation in human cancer.     

Role in tumorigenesis of silent mutations in the TP53 gene

Over 10,000 mutations in the TP53 suppressor gene have been recorded in the International Agency for Research on Cancer (IARC) tumor data base. About 4% of these mutations are silent. It is a question whether these mutations play a role in tumor development. In order to approach this question, we asked whether the reported silent mutations are randomly distributed throughout the TP53 gene. The p53 data base was searched exon by exon. From the frequency of codons with no silent mutations, the average number of silent mutations per codon for each exon was calculated using the Poisson distribution. The results indicate the distribution to be non-random. About one-third of all silent mutations occur in "hot-spots" and after subtraction of these hot-spots, the remaining silent mutations are randomly distributed. In addition, the percentage of silent mutations among the total in the silent mutation hot-spots is close to that expected for random mutation. We conclude that most of the silent mutations recorded in tumors play no role in tumor development and that the percentage of silent mutation is an indication of the amount of random mutation during tumorigenesis. Silent mutations occur to a significantly different extent in different tumor types. Tumors of the esophagus and colon have a low frequency of silent mutations, tumors of the prostate have a high frequency.     

Multiple mutations of the p53 gene in human mammary carcinoma

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.     

Missense mutations in cancer suppressor gene TP53 are colocalized with exonic splicing enhancers (ESEs)

Mutation databases can be viewed as footprints of functional organization of a gene and thus can be used to infer its functional organization. We studied the association of exonic splicing enhancers (ESEs) with missense mutations in the tumor suppressor gene TP53 using the International Agency for Research on Cancer (IARC) mutation database. The goals of the study were: (i) to verify the hypothesis that deleterious missense mutations are colocalized with ESEs; (ii) to identify potentially functional ESE sites in the open reading frame (ORF) of the TP53. If some sequence functions as a splicing enhancer, then nucleotide substitutions in the site will disturb splicing, abrogate p53 function, and cause an increased susceptibility to cancer. Therefore, among cancers showing p53 mutations, more missense mutations are expected within functional ESE sites as compared to non-functional ESE motifs. Using several statistical tests, we found that missense mutations in TP53 are strongly colocalized with ESEs, and that only a small fraction of ESE sites contributes to the association. There are usually one or two ESEs per exon showing a statistically significant association with missense mutations--so-called significant ESE sites. In many respects significant ESE sites are different from those that do not show association with missense mutations. We found that positions of significant ESE sites are codon-dependent--significant ESEs preferentially start from the first position of a codon, whereas non-significant ESEs show no position dependence. Significant ESEs showed a more limited set of sequences compared to non-significant ESEs. These findings suggest that there is a limited number of missense mutations that influence ESE sites and our analysis provides further insight into the types of sites that harbor exonic enhancer elements.     

Role in tumorigenesis of silent mutations in the TP53 gene

Over 10,000 mutations in the TP53 suppressor gene have been recorded in the International Agency for Research on Cancer (IARC) tumor data base. About 4% of these mutations are silent. It is a question whether these mutations play a role in tumor development. In order to approach this question, we asked whether the reported silent mutations are randomly distributed throughout the TP53 gene. The p53 data base was searched exon by exon. From the frequency of codons with no silent mutations, the average number of silent mutations per codon for each exon was calculated using the Poisson distribution. The results indicate the distribution to be non-random. About one-third of all silent mutations occur in “hot-spots” and after subtraction of these hot-spots, the remaining silent mutations are randomly distributed. In addition, the percentage of silent mutations among the total in the silent mutation hot-spots is close to that expected for random mutation. We conclude that most of the silent mutations recorded in tumors play no role in tumor development and that the percentage of silent mutation is an indication of the amount of random mutation during tumorigenesis. Silent mutations occur to a significantly different extent in different tumor types. Tumors of the esophagus and colon have a low frequency of silent mutations, tumors of the prostate have a high frequency.     

Spontaneous multiple mutations show both proximal spacing consistent with chronocoordinate events and alterations with p53-deficiency

Analysis of spontaneous multiple mutations in normal and tumor cells may constrain hypotheses about the mechanisms responsible for multiple mutations and provide insight into the mutator phenotype. In a previous study, spontaneous doublets in Big Blue mice were dramatically more frequent than expected by chance and exhibited a mutation pattern similar to that observed for single mutations [Mutat. Res. 452 (2000) 219]. The spacing between mutations in doublets was generally closer than expected by chance and the distribution of mutation spacing fit an exponential, albeit with substantial scatter. We now analyze 2658 additional mutants and confirm that doublets are enhanced dramatically relative to chance expectation. The spacing, frequency and pattern of spontaneous doublets and multiplets (domuplets) are examined as a function of age, tissue type, p53-deficiency and neoplasia in the new and combined data. The new and combined data confirm that the distribution of the spacing between mutations in doublets is non-random with the mutations more closely spaced than expected by chance (P < 0.0005; combined data), consistent with temporally coordinate (chronocoordinate) events. An exponential provides an excellent fit to the distribution (R2 = 0.98) and estimates that half of doublets have mutations separated by 120 nucleotides or less (the "half-life of mutation spacing"). We make several novel observations: (i) singlets and doublets show similar overall increases in frequency with age (ii) doublet frequency may be lower in the male germline, consistent with the generally reduced mutation frequency in the male germline (iii) doublet frequencies are elevated in somatic tissues of p53-deficient mice (Li-Fraumini cancer syndrome model; P = 0.005) and (iv) doublets and singlets in tumors from p53-deficient mice have a different mutation pattern (P = 0.007). The observations are consistent with chronocoordinate occurrence of spontaneous doublets and multiplets due to a transient error-prone condition and do not suggest a major role for the recently discovered Y family of error-prone polymerases. The enhancement of doublets in p53-deficient mice may contribute to cancer risk.     

Genomic features defining exonic variants that modulate splicing

Background  

Single point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative.     

Hearing silence: non-neutral evolution at synonymous sites in mammals

Although the assumption of the neutral theory of molecular evolution - that some classes of mutation have too small an effect on fitness to be affected by natural selection - seems intuitively reasonable, over the past few decades the theory has been in retreat. At least in species with large populations, even synonymous mutations in exons are not neutral. By contrast, in mammals, neutrality of these mutations is still commonly assumed. However, new evidence indicates that even some synonymous mutations are subject to constraint, often because they affect splicing and/or mRNA stability. This has implications for understanding disease, optimizing transgene design, detecting positive selection and estimating the mutation rate.     

A polymorphism but no mutations in the GADD45 gene in breast cancers

The p53 gene product is part of a pathway regulating growth arrest at the G₁ checkpoint of the cell cycle. Mutation of other components of this pathway, including the products of the ataxia telangiectasia (AT), GADD45, mdm2, and p21^WAF1/CIP1 genes may have effects comparable to mutations in the p53 gene. The GADD45 gene is induced by ionizing radiation and several DNA-damaging xenobiotics. Induction requires the binding of wild-type p53 to an evoulutionarily highly conserved putative intronic p53 binding site in intron 3 of GADD45. We recently analyzed the entire coding region of the p53 gene in primary breast cancers of Midwestern white women and found 21 mutations among 53 tumors (39,6%). We now have shown by direct sequencing that there are no mutations in the intronic p53 binding site of the GADD45 gene in any of the 53 primary breast cancers and no mutations in the entire coding region of the GADD45 gene in a subset of 26 consecutive tumors (12 with p53 mutation and 14 without p53 mutation). The only sequence variation detected was a common polymorphism in intron 3. The absence of mutations in the GADD45 gene, including the putative p53-binding intronic site, suggests that this gene is not a frequent target of mutations in breast cancer. Although mutations of the p53 gene have been studied in a wide spectrum of human cancers, GADD45 has not been examined in any tumor or cell line to the best of our knowledge. Our results raise the possibility that mutation of the GADD45 gene alone is not functionally equivalent to loss of wild-type p53 activity. Received: 14 September 1995     

Nucleotide polymorphism at the RpII215 gene in Drosophila subobscura. Weak selection on synonymous mutations

Nucleotide variation in an 8.1-kb fragment encompassing the RpII215 gene, which encodes the largest subunit of the RNA polymerase II complex, is analyzed in a sample of 11 chromosomes from a natural population of Drosophila subobscura. No amino acid polymorphism was detected among the 157 segregating sites. The observed numbers of preferred and unpreferred derived synonymous mutations can be explained by neutral mutational processes. In contrast, preferred mutations segregate at significantly higher frequency than unpreferred mutations, suggesting the action of natural selection. The polymorphism to divergence ratio is different for preferred and unpreferred changes, in agreement with their beneficial and deleterious effects on fitness, respectively. Preferred and unpreferred codons are nonrandomly distributed in the RpII215 gene, leading to a heterogeneous distribution of polymorphic to fixed synonymous differences across this coding region. This intragenic variation of the polymorphism/divergence ratio cannot be explained by different patterns of gene expression, mutation, or recombination rates, and therefore it indicates that selection coefficients for synonymous mutations can vary extensively across a coding region. The application of nucleotide composition stationarity tests in coding and flanking noncoding regions, assumed to behave neutrally, allows the detection of the action of natural selection when stationarity holds in the noncoding region.     

Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome.

Germ-line mutations of the tumor-suppressor gene p53 have been observed in some families with the Li-Fraumeni syndrome (LFS), a familial cancer syndrome in which affected relatives develop a diverse set of early-onset malignancies including breast carcinoma, sarcomas, and brain tumors. The analysis of the p53 gene in LFS families has been limited, in most studies to date, to the region between exon 5 and exon 9. In order to determine the frequency and distribution of germ-line p53 mutations in LFS, we sequenced the 10 coding exons of the p53 gene in lymphocytes and fibroblast cell lines derived from 15 families with the syndrome. Germ-line mutations were observed in eight families. Six mutations were missense mutations located between exons 5 and 8. One mutation was a nonsense mutation in exon 6, and one mutation was a splicing mutation in intron 4, generating aberrant shorter p53 RNA(s). In three families, a mutation of the p53 gene was observed in the fibroblast cell line derived from the proband. However, the mutation was not found in affected relatives in two families and in the blood from the one individual, indicating that the mutation probably occurred during cell culture in vitro. In four families, no mutation was observed. This study indicates that germ-line p53 mutations in LFS are mostly located between exons 5 and 8 and that approximately 50% of patients with LFS have no germ-line mutations in the coding region of the p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide polymorphism in the RpII215 gene region of the insular species Drosophila guanche: reduced efficacy of weak selection on synonymous variation

An approximately 6.9-kb region encompassing the RpII215 gene was sequenced for 24 individuals of the island endemic species Drosophila guanche. The comparative analysis of synonymous polymorphism and divergence in D. guanche and D. subobscura, two species with pronounced differences in population size, allows contrasting the nearly neutral character of synonymous mutations. In D. guanche, unlike in D. subobscura, (1) the ratio of preferred to unpreferred synonymous changes was similar for polymorphic and fixed changes, (2) the numbers of preferred and unpreferred changes, both polymorphic and fixed, could be explained by the mutational process, and (3) the estimated scaled selection coefficient for unpreferred mutations did not differ significantly from zero. Additionally, the comparative analysis revealed that both the ratio of preferred to unpreferred synonymous changes and the frequency spectrum of unpreferred polymorphic mutations differed significantly between species. All these results indicate that a large fraction of synonymous mutations in the RpII215 gene behave as effectively neutral in D. guanche, whereas they are weakly selected in D. subobscura. The reduced efficacy of selection in the insular species constitutes strong evidence of the nearly neutral character of synonymous mutations and, therefore, of the role of weak selection in maintaining codon bias.     

Mutations in the Trp53 gene of UV-irradiated Xpc mutant mice suggest a novel Xpc-dependent DNA repair process

Mutational hot spots in the human p53 gene are well established in tumors in the human population and are frequently negative prognosticators of the clinical outcome. We previously developed a mouse model of skin cancer with mutations in the xeroderma pigmentosum group C gene (Xpc). UVB radiation-induced skin cancer is significantly enhanced in these mice when they also carry a mutation in one copy of the Trp53 gene (Xpc-/-Trp53+/-). Skin tumors in these mice often contain inactivating mutations in the remaining Trp53 allele and we have previously reported a novel mutational hot spot at a non-dipyrimidine site (ACG) in codon 122 of the Trp53 gene in the tumors. Here we show that this mutation is not a hot spot in Xpa or Csa mutant mice. Furthermore, the mutation in codon T122 can be identified in mouse skin DNA from (Xpc-/-Trp53+/-) mice as early as 2 weeks after exposure to UVB radiation, well before histological evidence of dysplastic or neoplastic changes. Since this mutational hot spot is not at a dipyrimidine site and is apparently Xpc-specific, we suggest that some form of non-dipyrimidine base damage is normally repaired in a manner that is distinct from conventional nucleotide excision repair, but that requires XPC protein.     

Household Income Is Associated with the p53 Mutation Frequency in Human Breast Tumors

Background

A study from Scotland reported that the p53 mutation frequency in breast tumors is associated with socio-economic deprivation.

Methods

We analyzed the association of the tumor p53 mutational status with tumor characteristics, education, and self-reported annual household income (HI) among 173 breast cancer patients from the greater Baltimore area, United States.

Results

p53 mutational frequency was significantly associated with HI. Patients with < $15,000 HI had the highest p53 mutation frequency (21%), followed by the income group between $15,000 and $60,000 (18%), while those above $60,000 HI had the fewest mutations (5%). When dichotomized at $60,000, 26 out of 135 patients in the low income category had acquired a p53 mutation, while only 2 out of 38 with a high income carried a mutation (P < 0.05). In the adjusted logistic regression analysis with 3 income categories (trend test), the association between HI and p53 mutational status was independent of tumor characteristics, age, race/ethnicity, tobacco smoking and body mass. Further analyses revealed that HI may impact the p53 mutational frequency preferentially in patients who develop an estrogen receptor (ER)-negative disease. Within this group, 42% of the low income patients (< $15,000 HI) carried a mutation, followed by the middle income group (21%), while those above $60,000 HI did not carry mutations (P _trend < 0.05).

Conclusions:

HI is associated with the p53 mutational frequency in patients who develop an ER-negative disease. Furthermore, high income patients may acquire fewer p53 mutations than other patients, suggesting that lifetime exposures associated with socio-economic status may impact breast cancer biology.