Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.     

p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation

Forty-two patients with hepatocellular carcinoma (HCC) were resected and their tumours were analysed for p53 mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) and gene sequencing. All the exons have been analysed in this study. Eight of 12 HCCs with cirrhosis due to viral hepatitis and the two patients with sarcomatoid changes displayed p53 mutations. In contrast, no mutation was observed in the fibrolamellar variant (n = 9), non-cirrhotics (n = 13) and alcoholic cirrhosis (n = 6). The mutations observed were in exons 5-8. Two mutations were observed in codons 136 and 213 as well as a T insertion between residues 156 and 157 (exon 5) and these are reported for the first time in HCC. Likewise, the silent mutation polymorphism in codon 213 was noticed in 3 of the 42 patients. Survival analysis of these patients after surgery showed the mean and median survival in patients with wild-type p53 to be 60 and 43 months respectively. In the group with p53 mutations, the mean and median survival was 15 and 12 months. The difference was statistically significant (P= 0.003).     

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.     

P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens

BACKGROUND/AIMS: Mutations in p53, a tumor suppressor gene located on chromosome 17p, are the most frequent genetic alterations found in human cancers. Increased intracellular concentration of p53, which is frequently but not systematically related to p53 mutation, has been proposed to be associated with poor prognosis in some tumor types. In colorectal cancer, this significance is still a matter of debate. To directly investigate the relationship between prognosis and p53 mutation, this study screened a series of 85 colorectal carcinomas for mutations in exons 5-8 of this gene. METHODS: Polymerase chain reaction-amplified products from tumor DNA were analyzed by denaturing gradient gel electrophoresis and direct DNA sequencing. RESULTS: Forty-four tumors were found to be mutated (52%). A strong correlation between the presence of a mutation and short survival was observed (P = 0.003). When tumors were classified according to their histological stage, a multivariate Cox model analysis showed that p53 mutation, rather than 17p allelic loss (previously proposed to convey prognostic information), was retained as the only independent prognostic factor (relative risk, 2.25; 95% confidence interval, 1.06-4.80; P < 0.029). CONCLUSIONS: Combined with staging, direct monitoring of p53 mutation improves prognostic accuracy for colorectal cancer.     

Optimized detection of DNA point mutations by double gradient denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis displays the highest detection rate among mutation scanning methods. In classical denaturing gradient gel electrophoresis the denaturant gradient range and migration times vary for every amplicon to be scanned, greatly affecting the routine application of the method. As an alternative, we developed double gradient denaturing gradient gel electrophoresis where a gradient of pore size is superimposed over the denaturing one, allowing maintenance of the zone-sharpening effect even over prolonged time runs, and adoption of identical run time conditions for all fragments analyzed. Here double gradient denaturing gradient gel electrophoresis has been applied to the analysis of a number of point mutations and polymorphisms located in several exons of three different genes, the cystic fibrosis transmembrane conductance regulator, the beta-globin and the p53 genes.     

Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues

A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.     

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.     

Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model

Two-dimensional (2-D) gene scanning (TDGS) is a method for mutation detection based on the electrophoretic separation of PCR-amplified DNA fragments according to size and base pair sequence. The use of denaturing gradient gel electrophoresis (DGGE) as the second separation step provides virtually 100% sensitivity, while the 2-D format allows the inspection of multiple gene fragments simultaneously. Analysis of many exons in parallel is greatly facilitated by extensive PCR multiplexing based on preamplification by long-distance PCR. Recently, TDGS has been applied to detect mutations in the retinoblastoma tumor suppressor gene RB1. Using RB1 as a model, we have now analyzed each step of the protocol, presenting overall improvements and a detailed cost analysis, where the total cost of the assay is found to be about $40 (US). An overall picture of TDGS cost-performance, as compared to direct sequencing, is provided as a function of the number of target fragments.     

Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis

Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a "GC-clamp" is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.     

Recovery of respiratory parameters and oxygen consumption during the recovery phase of cardiopulmonary exercise test in patients with dilated cardiomyopathy]

Cowden syndrome (CS) or multiple hamartoma syndrome (MIM 158350) is an autosomal dominant disorder with an increased risk for breast and thyroid carcinoma. The diagnosis of CS, as operationally defined by the International Cowden Consortium, is made when a patient, or family, has a combination of pathognomonic major and/or minor criteria. The CS gene has recently been identified as PTEN, which maps at 10q23.3 and encodes a dual specificity phosphatase. PTEN appears to function as a tumour suppressor in CS, with between 13-80% of CS families harbouring germline nonsense, missense, and frameshift mutations predicted to disrupt normal PTEN function. To date, only a small number of tumour suppressor genes, including BRCA1, BRCA2, and p53, have been associated with familial breast or breast/ovarian cancer families. Given the involvement of PTEN in CS, we postulated that PTEN was a likely candidate to play a role in families with a "CS-like" phenotype, but not classical CS. To answer these questions, we gathered a series of patients from families who had features reminiscent of CS but did not meet the Consortium Criteria. Using a combination of denaturing gradient gel electrophoresis (DGGE), temporal temperature gel electrophoresis (TTGE), and sequence analysis, we screened 64 unrelated CS-like subjects for germline mutations in PTEN. A single male with follicular thyroid carcinoma from one of these 64 (2%) CS-like families harboured a germline point mutation, c.209T-->C. This mutation occurred at the last nucleotide of exon 3 and within a region homologous to the cytoskeletal proteins tensin and auxilin. We conclude that germline PTEN mutations play a relatively minor role in CS-like families. In addition, our data would suggest that, for the most part, the strict International Cowden Consortium operational diagnostic criteria for CS are quite robust and should remain in place.     

Solvent polarity and pH effects on the spectroscopic properties of neutral red: application to lysosomal microenvironment probing in living cells

To investigate the molecular mechanism of gastric carcinogenesis, we analyzed genetic instability and p53 gene mutations in 40 primary gastric carcinomas. Tumor samples were from untreated patients with no family history suggestive of genetic predisposition to cancer. We screened six microsatellite loci by the polymerase chain reaction (PCR) method, and exons 5-8 of the p53 gene by the PCR-based denaturing gradient gel electrophoresis and sequencing techniques. Microsatellite instability was detected in 32.5% (13/40), and gene mutations in 40% (16/40), of the tumors analyzed. No statistically significant associations were found between genetic alterations and clinico-pathological variables (with the exception of diffusion of lymph node metastases, which was inversely associated with the presence of microsatellite alterations; P < 0.01). Interestingly, a negative association was found between genetic instability and p53 gene mutations: 11 out of 13 tumors showing instability proved to carry a nonmutated p53 gene versus 2/13 carrying a mutated gene (P = 0.03). These observations suggest that genetic instability and p53 gene mutations play a crucial role in the gastric carcinogenic process, but likely act through distinct pathways during cancer development. However, genetic instability is not in and of itself neoplastic. Therefore, we investigated whether insertion/deletion mutations of the polyadenine tract within the transforming growth factor-beta type II receptor gene (TGF-beta RII) were frequently present in gastric tumors with an RER+ (replication error) phenotype. We found RII mutations in 8/40 (20%) samples: mutations were present in 7/13 (54%) RER+ tumors versus 1/27 (4%) RER- cases (P < 0.001).     

Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.     

Analysis of genetic disorders of cancer of the rectum: differences in relation to cancer of the colon

AIMS AND METHODS: We studied the mechanisms of colon and rectal carcinogenesis by analysing in a series of 83 rectal tumors the prevalence of the two tumor types characteristic of colon cancer, i.e., the LOH+ type, defined by p53 and APC mutations (studied by DGGE and protein truncation assay), and the RER+ type, which is characterized by the instability of some mononucleotide repeat microsatellites (Bat 25 and Bat 26). Additionally, we analyzed the occurrence of Ki-Ras mutations (direct sequencing). RESULTS: Only one tumor turned out to be RER+. Moreover, in 59% of the tumor cases mutations were found in p53, essentially affecting codon 175. The APC and Ki-Ras genes were found to be mutated in 40 and 26% of the rectal tumors, respectively. In 18 tumors (21%) none of the genes studied were mutated. CONCLUSIONS: The RER+ phenotype is rare among rectal tumors, which are essentially LOH+. In these LOH+ tumors the p53 gene is more frequently mutated than in colorectal tumors with the same phenotype. Mutations in the APC and Ki-Ras genes, on the other hand, are less frequent in rectal tumors. Tumors with the RER- and LOH- phenotype may develop as a result of a third carcinogenesis model which must be defined.     

Molecular analysis of tumor suppressor genes p16INK4 and TP53 of osteosarcomas in Spanish children

OBJECTIVE: Alterations affecting tumor suppressor genes, specifically p16INK4 and TP53, have been shown to be involved in the development of human cancer due to their important role in the control of normal cell cycle progression. As the genetic events leading to the development of pediatric osteosarcoma remain partially unclear, we have tested the possibility that a significant number of pediatric osteosarcoma patients harbor mutations in these genes. PATIENTS AND METHODS: We have analyzed 64 samples (fresh tissues, paraffin embedded biopsies and peripheral blood lymphocytes) corresponding to 38 pediatric osteosarcoma patients. TP53 mutations were analyzed by DGGE (Denaturing Gradient Gel Electrophoresis) analysis of exons 5 through 8. We searched for deletions in the p16INK4 gene by PCR (Polymerase Chain Reaction) analysis and point mutations were screened by means of SSCP (Single Strand Conformation Polymorphisms). RESULTS: Our analysis showed that 18.4% of the samples harbored mutations in the coding region of TP53 and that 7% had a homozygous deletion of the p16INK4 gene. Our results suggest that p16INK4 deletions may constitute a bad prognostic factor and that TP53 alterations may be correlated, although not statistically, with reduced survival time. CONCLUSIONS: Mutations of the TP53 and deletion of p16INK4 tumor suppressor genes seem to be involved in the development of pediatric osteosarcoma. Moreover, alterations of these genes may constitute a prognostic factor related with poor prognosis or decreased survival time.     

Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas

Glioblastoma multiforme, the most malignant human brain tumor, may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). We present further evidence that primary and secondary glioblastomas constitute distinct disease entities which develop through the acquisition of different genetic alterations. We analyzed p53 mutations, p53 protein accumulation and epidermal growth factor receptor (EGFR) overexpression in 49 biopsies classified as primary or secondary glioblastoma according to clinical and histopathologic criteria. Patients with primary glioblastoma were selected on the basis of a clinical history of less than 3 months and histopathologic features of glioblastoma at the first biopsy (19 cases; mean age, 55 years). The diagnosis of secondary glioblastomas required at least two biopsies and clinical as well as histologic evidence of progression from low grade or anaplastic astrocytoma (30 cases; mean age, 39 years). DNA sequence analysis showed that p53 mutations were rare in primary glioblastomas (11%) while secondary glioblastomas had a high incidence of p53 mutations (67%), of which 90% were already present in the first biopsy. The incidence of p53 protein accumulation (nuclear immunoreactivity to PAb 1801) was also lower in primary (37%) than in secondary glioblastomas (97%). In contrast, immunoreactivity for the EGF receptor prevailed in primary glioblastomas (63%) but was rare in secondary glioblastomas (10%). Only one out of 49 glioblastomas showed EGFR overexpression and a p53 mutation. These data indicate that overexpression of the EGF receptor and mutations of the p53 tumor suppressor gene are mutually exclusive events defining two different genetic pathways in the evolution of glioblastoma as the common phenotypic endpoint.     

Altered WAF1 genes do not play a role in abnormal cell cycle regulation in breast cancers lacking p53 mutations

Seventy-five to 80% of breast cancers are negative for p53 gene mutations. We have investigated the possibility that altered WAF1 genes provide an alternative mode of cell cycle disruption in these tumors. DNA from a total of 85 primary breast tumors and cell lines from both the United States and Australia were examined for WAF1 and p53 mutations. With the exception of one primary tumor containing the polymorphic codon 31 (AGC-->AGA), no missense mutations in the WAF1 gene were found in 33 primary tumors or in the 19 cell lines from the United States. By contrast, 2 of 33 tumors from Australia contained tumor-specific missense mutations in the WAF1 gene, while an additional six cases contained the AGC-->AGA polymorphic 31st codon in the WAF1 gene. The p53 mutation frequency in the Australian cohort (18%) was found to be similar to that reported by us (Glebov et al., Cancer Res., 54: 3703-3709, 1994; Runnebaum et al., Proc. Natl. Acad. Sci. USA, 88: 10657-10661, 1991) in the tumors of United States patients (13%) with sporadic breast cancer. Thus, mutations in the WAF1 gene are rare in tumors with or without p53 mutations, suggesting that except in a minor population of breast cancer patients of Caucasian origin, cell cycle dysregulation by mutated p53 or WAF1 genes may not contribute to breast tumor initiation or progression.     

Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.     

A novel mutation of presenilin 1 in familial Alzheimer's disease in Israel detected by denaturing gradient gel electrophoresis

Germline mutations in the presenilin 1 (PS1) gene apparently account for the majority of early-onset, familial Alzheimer's disease (AD). Using a mutation-screening strategy (denaturing gradient gel electrophoresis; DGGE), we analyzed a large family with early onset AD and seizures. The patients in this family showed a novel missense mutation in exon 5 of the PS1 gene (A to T change in codon 120, altering glutamine to aspartic acid). This novel mutation is located within the second hydrophilic domain of the molecule, a region not particularly involved in previously described germline mutations, and is of unknown biological significance. These results also demonstrate that DGGE can be used effectively to screen for mutations within this gene.     

Five novel mutations of the protein S active gene (PROS 1) in 8 Norman families

To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturing gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus' gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gln mutation.     

Molecular epidemiology of breast cancers in northern and southern Japan: the frequency, clustering, and patterns of p53 gene mutations differ among these two low-risk populations

Comparison of acquired mutations in the p53 tumor suppressor gene can illuminate factors contributing to carcinogenesis among cancer cohorts. Japan has an ethnically homogeneous population with a low incidence of breast cancer. Previously we reported an unusual frequency, allelic status, and clustering of mutations in breast cancers from the northern part of the main Japanese island. To extend these findings, exons 2-11 and adjacent intronic sequences were analysed in tumors of women from northern (Hokkaido) and southern (Tokushima) Japan. The frequency of breast cancers with p53 gene mutations in the Hokkaido group is the highest reported (81%) while that in Tokushima (28%) is similar to most other populations. Thirteen of the 19 mutations (68.4%) in the Hokkaido cohort were heterozygous, an unusually high frequency for p53 mutations in any tumor type. There were three missense mutations at codon 175, a known hotspot for alterations in the p53 gene, and three missense mutations at codon 179, a rare site for p53 changes. In addition, the patterns of p53 gene mutation differed between the two Japanese cohorts (P=0.04). The multiple differences in acquired p53 mutations suggest unsuspected biological differences among breast cancers in northern and southern Japan. In addition, the high frequency of p53 mutations in breast cancers from Hokkaido predicted a poorer prognosis for this population which was confirmed on examination of mortality data.