Germline P53 Mutations Research Articles

Genomic instability due to germline p53 mutations drives preneoplastic Progression toward cancer in human cells

Cells heterozygous for mutations in p53 demonstrate extreme genomic instability and develop mutations detectable at the chromosome level as well as the molecular level. This genomic instability causes initially nontumorigenic ras-expressing immortal LFS cells to progress to a tumorigenic state presumably due to additional mutational events. It is not surprising that LFS families with these p53 mutations develop the additional mutations necessary for cancer to occur at such high frequencies. This observation is consistent with increased cancer rates in these families being due to abrogation of a rate limiting step rather than a rate expected for one less step in a multistep carcinogenic process. Although p53 has been shown to be able to function as a transcription factor, mutations in p53 appear to affect genomic stability in LFS fibroblasts with double minutes and telomeric associations being prominent early events. One possibility is that p53 controls the expression of genes required for fidelity of replication or telomerase activity. Alternatively p53 may itself be a replication factor like the transcription factor CTF. In the future, we plan to investigate whether p53 plays a direct role in replication.

ARCAD: A method for estimating age‐dependent disease risk associated with mutation carrier status from family data

We present ARCAD, a method to estimate the disease risk associated with mutation carrier status using data on families ascertained by affected individuals, in which a germline mutation has been detected. Because the event of interest, the age of onset, is a censored variable, the method uses the survival analysis approach to formulate the likelihood. Provided that selection criteria are clearly defined, the ascertainment bias is removed by including a correction term in the likelihood computation. We simulated family data and selected those with a proband affected before age 17, and at least one or at least two relatives affected before age 46. We show that including the correction for the ascertainment provides reliable estimates of the risk, even when many individuals are not tested for the mutation. An application to cancer risk and germline p53 mutations is presented. We routinely investigate the p53 status for all the children treated in the Department of Pediatric Oncology at the Institute Gustave Roussy, whose family displays at least one relative affected by cancer before age 46. We identified 5 families with an inherited germline p53 mutation. The risk for any cancer for a mutation carrier estimated by ARCAD was 42% within the age class 0-16 years, 38% within the age class 17-45 years, and 63% after 45 years, with a lifetime risk of 85%. These risks are almost entirely explained by the occurrence of the six most frequent cancers encountered in the Li-Fraumeni syndrome.

Germline p53 mutations are frequently detected in young children with rhabdomyosarcoma.

We investigated the possibility that a proportion of children with sporadic rhabdomyosarcoma (RMS) carry constitutional mutations of the p53 tumor suppressor gene. 33 patients with sporadic RMS at two large outpatient pediatric oncology clinics submitted blood samples. Genomic DNA was extracted from peripheral blood leukocytes and PCR was used to amplify exons 2-11 of the p53 gene. Amplified genomic DNA was screened for the presence of germline p53 mutations using single-strand conformation polymorphism (SSCP) analysis. The DNA sequence of those samples that showed aberrant migration of bands on SSCP analysis was determined to identify the precise nature of the gene mutations. Patient records were reviewed to assess clinical correlates of the mutant p53 carrier state. Heterozygous constitutional mutations were detected in 3/33 patient samples screened. Two of these missense mutations are located in exon 7 and one in exon 8 of the p53 gene. The presence of mutations was not correlated with tumor histology, stage, or site. However, an association between young age at diagnosis and presence of a constitutional p53 mutation was noted: 3/13 children under the age of 3 yr at diagnosis carried mutations, whereas none of 20 children over 3 yr of age at diagnosis harbored a detectable constitutional mutation. These results in children with RMS corroborates previous findings in other clinical settings suggesting that the mutant p53 carrier state may predispose individuals to malignancy at an early age. Although this study did not assess whether the mutations were preexisting or new germline alterations, assessment of close relatives of RMS patients for cancer risk and predictive genetic testing may be indicated.

P53 polymorphisms and haplotypes in different ethnic groups.

Three different p53 polymorphisms (a codon 72 BstUI RFLP, a 16-bp duplication in intron 3 and an MspI RFLP in intron 6) and haplotype combinations were studied in three northern European populations, viz. Finns, Swedes and Swedish Saamis. Significant ethnic differences were found in the codon 72 and 16-bp polymorphisms. The three polymorphisms were in strong linkage disequilibria, all of which were highly significant (p < 1 x 10(-18)), and ethnic differences with respect to linkage disequilibria were observed. Estimates of the frequencies of extended haplotypes showed that the most common ('wild-type') haplotype in the three populations was 2-1-2, viz, the codon 72 Arg allele linked to absence of the 16-bp duplication and presence of the MspI site. There were two additional common haplotypes, 1-1-2 and 1-2-1, and five rare haplotypes with a combined frequency of about 0.03. The present results indicate that extended haplotypes would be more informative in studies of population differences and associations between p53 germline mutations and cancer.

Bilateral Breast Cancer in a Patient with a Strong Family History of Cancer: Analysis of p53 Germ Line Mutations.

We report a case of bilateral breast carcinoma in a patient with a strong family history, including 4 cases of breast carcinoma, 1 case of prostate carcinoma (father), 1 case of hepatocellular carcinoma (mother), 2 cases of gastric carcinoma, 1 case of lung carcinoma, and 1 case of lingual carcinoma, in second degree relatives, together with analysis of germ line p53 mutations. The patient was a 51-year-old female who had undergone mastectomy 9 years previously for an invasive ductal carcinoma of the right breast. Lymph nodes were free of metastases and the tumor had negative estrogen receptor (ER) status. Bone and lung metastases developed 18 months after surgery, and had been well controlled with chemoendocrine therapy. She subsequently underwent a modified radical mastectomy for carcinoma in the contralateral breast. This was an invasive lobular carcinoma with negative lymph node metastasis, negative p53 immunoreaction, negative c-erbB-2 protein and positive ER status. In this breast-prostate carcinoma-type cancer family there was a high incidence of breast carcinoma; the father, who had prostate carcinoma, was possibly a carrier of a breast carcinoma susceptible gene. We have however detected to p53 germ line mutations in the lymphocytes DNA of the patient and her niece. The accumulation of cancers in this family line remains to be elucidated further using other genetic markers.

Germline p53 mutations and heritable cancer.

Germline p53 mutations and heritable cancer.

High frequency of germline p53 mutations in childhood adrenocortical cancer.

Adrenocortical carcinoma (ADCC) is a rare childhood cancer, affecting three of 1 million children younger than 16 years old in the United States. ADCC may be found in association with the Li-Fraumeni and Beckwith-Wiedemann syndromes. Children with ADCC are also at substantially increased risk of second primary cancers. Because of these associations, it is believed that the genetic basis for ADCC is stronger than for most childhood malignancies. Germline mutations of the TP53 tumor suppressor gene are associated with cancer predisposition in families with the Li-Fraumeni syndrome as well as in individuals with sporadically occurring component tumors of the syndrome. We investigated the possibility that germline TP53 gene alterations existed in children with ADCC. Sixteen children with ADCC under the age of 18 were identified from searches of medial oncology records at three Canadian hospitals. Eleven of these 16 patients identified were alive. The mean age at diagnosis was 4.8 years (range, 1-17 years). Family histories were obtained for 11 unselected children with ADCC (six girls and five boys). Pathologic confirmation of tumor diagnosis was obtained from the medical records. Using single-strand conformational polymorphism analysis followed by single-strand DNA sequencing, genomic DNA extracted from whole blood was analyzed for the presence of TP53 mutations for six living ADCC patients. Three of six (50%) children were found to carry germline TP53 mutations in exons 5, 6, and 7, respectively. Both wild-type and mutant alleles were identified in all three TP53 sequences, indicating that the patients were heterozygous for germline TP53 mutations. None of these children was from a family with the Li-Fraumeni syndrome. The mutation in one child was shown to be inherited from the mother, who subsequently developed breast cancer. A striking excess of cancer was found in one family of a patient carrying wild-type TP53. Our observation of a high frequency of germline TP53 mutations in children with sporadic ADCC suggests that these children may represent probands with which to ascertain Li-Fraumeni syndrome families. It may be reasonable for children with adrenocortical carcinoma to be candidates for germline TP53 analysis. In light of the wealth of information in the Li-Fraumeni literature that associates germline TP53 mutations with a variety of malignancies, this testing may have important consequences for risk assessment for other close relatives, including early-onset breast cancer in the mothers.

Lack of germ-line mutations in the p53 gene exons 4 to 8 in patients with late-onset second malignant neoplasms

Previous studies have shown p53 germ-line mutations in some familial cancer aggregations with or without the Li-Fraumeni syndrome (LFS). Such mutations were also reported in children and young adults with second malignant neoplasms (SMN). This led us to screen for p53 germ-line mutations in a group of seven patients affected with SMN, but characterized by an older age of onset than in the previous reports. No mutation was found in exons 4 to 8 and their boundaries using the single-strand conformation polymorphism technique. Our results give strong evidence for genetic heterogeneity of SMN, probably related to the age of cancer onset.

Cooperative tumorigenic effects of germline mutations in Rb and p53

The tumour suppressor genes Rb and p53 are mutated in several types of human cancer, and many tumour types carry mutations in both genes. To study how these genes normally function, we and others have created mouse strains with Rb and p53 mutations. Here we describe the phenotypic effects of combined germline mutations in these two tumour suppressor genes. Mice mutant for both genes have reduced viability and exhibit novel pathology including pinealoblastomas, islet cell tumours, bronchial epithelial hyperplasia and retinal dysplasia. These data indicate that mutations in Rb and p53 can cooperate in the transformation of certain cell types in the mouse.

Cowden syndrome and Lhermitte-Duclos disease in a family: a single genetic syndrome with pleiotropy?

Cowden syndrome is an autosomal dominant condition of multiple hamartomas. Patients with this phakomatosis have an increased risk of breast cancer and thyroid tumours. Lhermitte-Duclos disease is usually a sporadic condition of cerebellar ganglion cell hypertrophy, ataxia, mental retardation, and self-limited seizure disorder. We describe a three generation family with Cowden syndrome and Lhermitte-Duclos disease. Karyotyping performed on the peripheral lymphocytes of the proband and her affected mother showed a 46,XX complement. Single strand conformational polymorphism analysis failed to show any germline p53 mutations as a cause of the syndrome in this family.

Germline mutations of the p53 tumor suppressor gene in children with osteosarcoma.

We investigated the possibility that a significant proportion of children with osteosarcoma harbor germline mutations of the p53 tumor suppressor gene and, therefore, this subgroup of pediatric cancer patients should be considered for large-scale predictive testing. Genomic DNA extracted from peripheral-blood leukocytes from 235 unselected children with osteosarcoma from 33 institutions were screened for the presence of germline p53 mutations using constant denaturant gel electrophoresis (CDGE). Exons 5 through 8 were evaluated in all patients and exon 2 and exon 9 were analyzed in 59 and 95 patients, respectively. Those samples that showed aberrant migration on CDGE were sequenced or analyzed by restriction enzyme digestion of polymerase chain reaction (PCR) products to confirm the nature of the gene alteration. In 18 samples, CDGE showed fragments of the p53 gene with altered electrophoretic mobilities compared with wild-type p53. DNA sequencing showed that 11 samples had an identical, previously described polymorphism. The other seven contained heterozygous p53 mutations located in exon 5 (n = 3), exon 6 (n = 1), exon 7 (n = 1), and exon 8 (n = 2). Six alterations were missense mutations and one was a nonsense mutation. Three of these patients had first-degree relatives with cancer. One of these three kindreds had a family history consistent with Li-Fraumeni syndrome (LFS). We identified germline p53 mutations in seven of 235 (3.0%) children with osteosarcoma. Four of these mutations were found in patients who did not have first-degree relatives with cancer. Although genetic transmission of the altered p53 gene could not be tested in this survey because of how it was designed, it is possible that predictive testing for p53 mutations could identify unaffected relatives of gene carriers who also have a high risk for the development of cancer. This study provides evidence for the importance of considering children with osteosarcoma for predictive testing for germline p53 mutations.

Tumor spectrum analysis in p53-mutant mice

Background: The p53 tumor suppressor gene is mutated in a large percentage of human malignancies, including tumors of the colon, breast, lung and brain. Individuals who inherit one mutant allele of p53 are susceptible to a wide range of tumor types. The gene encodes a transcriptional regulator that may function in the cellular response to DNA damage. The construction of mouse strains carrying germline mutations of p53 facilitates analysis of the function of p53 in normal cells and tumorigenesis.Results In order to study the effects of p53 mutation in vivo, we have constructed a mouse strain carrying a germline disruption of the gene. This mutation removes approximately 40 % of the coding capacity of p53 and completely eliminates synthesis of p53 protein. As observed previously for a different germline mutation of p53, animals homozygous for this p53 deletion mutation are viable but highly predisposed to malignancy. Heterozygous animals also have an increased cancer risk, although the distribution of tumor types in these animals differs from that in homozygous mutants. In most cases, tumorigenesis in heterozygous animals is accompanied by loss of the wild-type p53 allele.Conclusion We reaffirm that p53 function is not required for normal mouse development and conclude that p53 status can strongly influence tumor latency and tissue distribution.

Germline p53 Mutations and Heritable Cancer

Germline p53 Mutations and Heritable Cancer

Tumorigenic and developmental effects of combined germ-line mutations in Rb and p53.

Tumorigenic and developmental effects of combined germ-line mutations in Rb and p53.

Secondary breast cancer in patients presenting with osteosarcoma: Possible involvement of germline p53 mutations

Second malignancies following treatment for osteosarcoma are unusual. Breast cancer occurring in patients with osteosarcoma has been reported following therapeutic chest irradiation. We now report three cases of breast cancer occurring in young women who were successfully treated for osteosarcoma. These women had not received therapeutic chest irradiation and in two of the three women there was no family history of breast cancer. Peripheral blood was available for study from one case. Of import, this case demonstrated a germline mutation in exon 7 of the tumor suppressor gene, p53. The mutation was detected by constant denaturing gradient gel electrophoresis and confirmed by DNA sequencing. In this particular patient, inactivation of the p53 gene may be involved in the development of both the first and second malignancy.

A novel P53 germline mutation associated with a familial brain tumour syndrome

A novel P53 germline mutation associated with a familial brain tumour syndrome

Familial Ovarian Cancer

Familial cancer clusters provide a unique opportunity to elucidate the molecular mechanisms central to the development of malignancy. We have identified four families in which 11 members developed epithelial ovarian cancer. The clinical course of disease in these individuals with familial ovarian cancer appears to be very different from that of patients with nonfamilial ovarian cancer. In order to compare these disease states, 34 cases matched for age at diagnosis (57 years), tumor histology (all adenocarcinomas), and a preponderance of advanced grade and FIGO stage of disease were selected. Patients with familial ovarian cancer exhibited a 67% 5-year survival in comparison with a 17% 5-year survival in the nonfamilial ovarian cancer cases. Preliminary studies indicate a lack of overexpression of the HER-2/neu oncogene in the familial cancer members' tumors. This may correlate with the indolent character of their disease. Abnormal p53 tumor suppressor gene expression was noted in four of six cancers tested. We also found germ line p53 mutations in three of the four families, but neither the germ line or tumor p53 prevalence was 100%. This is the first report of germ line p53 mutations associated with familial ovarian cancer and indicates that this gene may play a role in the development of some familial ovarian cancers.

Frequency of p53 Tumor Suppressor Gene Mutations in Human Primary Brain Tumors

Mutations in the p53 tumor suppressor gene are the most common genetic alterations found in diverse types of human cancer, including the primary malignant brain tumor, glioblastoma multiforme. To estimate the frequency of p53 mutations in human brain tumors, we screened 120 human primary brain tumors (59 astrocytic; 61 nonastrocytic) by the polymerase chain reaction-single-strand conformation polymorphism technique. Six astrocytic tumors (one anaplastic astrocytoma and five glioblastoma multiforme) were found to have putative p53 mutations. Direct sequencing of polymerase chain reaction-amplified deoxyribonucleic acid from these six tumors confirmed the presence of different point mutations in the conserved regions of the p53 gene. Allelic losses on chromosome 17p were detected in four (67%) of the six tumors with p53 mutations. p53 mutations were not detected in any of the 61 nonastrocytic brain tumors. Also, polymerase chain reaction-single-strand conformation polymorphism analysis of 74 leukocyte deoxyribonucleic acid samples from patients with astrocytic and nonastrocytic brain tumors failed to detect any germ-line p53 mutations. We conclude from these findings that p53 gene mutations in brain neoplasms are primarily limited to tumors of astrocytic origin and that the p53 gene mutations in sporadic astrocytomas are somatic in origin (i.e., nonprenatally determined).

Screening patients for heterozygous p53 mutations using a functional assay in yeast

Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

Spectrum of Mutation and Frequency of Allelic Deletion of the p53 Gene in Ovarian Cancer

The p53 gene encodes a nuclear phosphoprotein present in low levels in normal human cells. The wild-type form of this protein functions to restrain inappropriate cellular proliferation. Approximately one half of human epithelial ovarian cancers have mutations in the p53 gene and overexpress the mutant protein product. Deletion of one allele of the p53 gene also frequently occurs in these cancers. We sought to define the spectrum of mutations in the p53 gene in epithelial ovarian cancer with respect to both the specific codons involved and the type of mutations observed. We also examined the frequency of allelic deletion of the p53 gene in cancers containing p53 gene mutations. Tissue samples from the epithelial ovarian cancers of 62 patients were obtained during initial laparotomy. Histologic examination was done to ensure that the experimental samples used in this study contained more than 75% cancer cells. Total RNA was extracted from these samples and separately from matched control noncancerous regions of the surgical specimen or white blood cells. The purified RNAs were reverse transcribed to generate cDNA copies of exons 4-10 of the p53 gene. Two rounds of polymerase chain reaction (PCR) were conducted to produce enough template for DNA sequence analysis of the regions of interest within the p53 gene. Dideoxy sequencing of at least two independent productions of each amplified DNA template was done to confirm the validity of the mutations found. Allelic deletions were identified by PCR and gel electrophoretic techniques to examine three polymorphisms within the p53 gene in cancer-normal DNA pairs. We identified 45 mutations in exons 5-8 of the p53 gene, where mutations frequently have been found in other cancer types. An additional mutation was identified in exon 4. Overall, 72% of the mutations were transitions, 24% transversions, and 4% microdeletions. Allelic deletion of the other p53 allele was seen in 67% of ovarian cancers in which a p53 mutation was present. Germ-line p53 mutations were not found in any patients whose cancers had p53 mutations. Like p53 mutations in other types of human cancers, those in epithelial ovarian cancers are diverse and occur frequently in exons 5-8. The predominance of transition mutations suggests that p53 mutations in ovarian cancer arise because of spontaneous errors in DNA synthesis and repair rather than the direct interaction of carcinogens with DNA. These molecular data are consistent with data from epidemiologic studies that have failed to demonstrate a convincing relationship between exposure to environmental carcinogens and the development of ovarian cancer.