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)     

Single base pair germ-line deletion in the p53 gene in a cancer predisposed family

A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Because this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppressor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated denaturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitutional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the formation of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.     

A database of germline p53 mutations in cancer-prone families.

We created a comprehensive database covering all published cases of germline p53 mutations. The current version lists 580 tumours in 448 individuals belonging to 122 independent pedigrees. The database describes each p53 mutation (type of the mutation, exon and codon affected by the mutation, nucleotide and amino acid change), each family (family history of cancer, diagnosis of Li-Fraumeni syndrome), each affected individual (sex, generation, p53 status, from which parent the mutation was inherited) and each tumour (type, age of onset, p53 status-loss of heterozygosity, immunostaining). Each entry contains the original reference(s). The database is freely available and can be obtained from http://www.lf2.cuni.cz     

Absence of p53 germ-line mutations in bilateral breast cancer patients

Summary The cause of Li-Fraumeni syndrome, a rare group syndrome of familial cancers, has recently been identified. Patients with this inherited condition are highly susceptible to specific neoplasms, including early-onset breast cancers. The available evidence links Li-Fraumeni syndrome to inherited mutations of the tumor suppressor gene p53. Moreover, somatically acquired p53 mutations and gene deletions are common feature in breast cancer of sporadic origin. These findings suggest that germline p53 mutations are important in familial and, possibly sporadic, breast tumors. We have therefore screened lymphocyte DNA from 19 unrelated bilateral cancer patients for germline p53 mutations in exons 5, 6, 7 and 8. We have however detected no germline mutations by means of the single-strand confirmation polymorphism technique in any of the lymphocyte DNAs examined and conclude that p53 mutations are not generally involved in bilateral breast cancer.     

Identification of a novel EYA1 splice-site mutation in a Danish branchio-oto-renal syndrome family

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by variable clinical manifestations including branchial fistulae, preauricular pits, ear malformations, hearing impairment, and renal anomalies. BOR is caused by mutations in the genes EYA1 and SIX1. A Danish BOR family with five affected individuals in three generations was analyzed for mutations in all 17 exons of EYA1 using direct sequencing of polymerase chain reaction (PCR) amplified genomic DNA. A novel splice-site mutation (IVS9+1 G>C) was detected in all affected family members but not in unaffected family members or in 96 controls. We conclude that this mutation is causing BOR in the family, most likely as a result of haploinsufficiency or an abnormal protein product caused by aberrant splicing of EYA1 mRNA.     

The mutational spectra of gene p53 in different types of tumors]

The comparative analysis of the frequencies of nucleotide exchanges in mutational spectra of gene p53 (5-8 exons) between germline cancer-prone families (Li-Fraumeni syndrome), between somatic mutations in the tumors of different histogenesis and cell lines, obtained from them, was carried out. The nucleotide positions with high level of mutation events (mutational "hot spots"), typical for germline mutational spectra, tissue-specific patterns of their disappearing and appearing of new ones in solid tumors in vivo, nearly complete absence of "hot spots" in lymphomas and cell lines in vitro were revealed. The obtained results allowed to suggest, that one of the leading factor controlling hot-spots distribution in 5-8 exons of gene p53 is the specificity of cell division conditions in vivo and in vitro.     

Germ-line mosaicism in tuberous sclerosis: how common?

Two-thirds of cases of tuberous sclerosis complex (TSC) are sporadic and usually are attributed to new mutations, but unaffected parents sometimes have more than one affected child. We sought to determine how many of these cases represent germ-line mosaicism, as has been reported for other genetic diseases. In our sample of 120 families with TSC, 7 families had two affected children and clinically unaffected parents. These families were tested for mutations in the TSC1 and TSC2 genes, by Southern blotting and by single-strand conformational analysis. Unique variants were detected in six families. Each variant was present and identical in both affected children of a family but was absent in both parents and the unaffected siblings. Sequencing of the variants yielded two frameshift mutations, one missense mutation, and two nonsense mutations in TSC2 and one nonsense mutation in TSC1. To determine which parent contributed the affected gametes, the families were analyzed for linkage to TSC1 and TSC2, by construction of haplotypes with markers flanking the two genes. Linkage analysis and loss-of-heterozygosity studies indicated maternal origin in three families, paternal origin in one family, and either being possible in two families. To evaluate the possibility of low-level somatic mosaicism for TSC, DNA from lymphocytes of members of the six families were tested by allele-specific PCR. In all the families, the mutant allele was detected only in the known affected individuals. We conclude that germ-line mosaicism was present in five families with mutations in the TSC2 gene and in one family with the causative mutation in the TSC1 gene. The results have implications for genetic counseling of families with seemingly sporadic TSC.     

Li-Fraumeni Syndrome: a p53 Family Affair

The p53 alterations frequently found in human tumors are missense mutations in the DNA binding domain. These p53 mutations have been shown to have gain-of-function or dominant negative properties in multiple experiments. The consequences of these p53 mutations at physiological levels on the development of a tumor were unclear. Using mouse models, three recent papers have shed light on the mechanisms of mutant p53 and its family members, p63 and p73, in tumorigenesis. Interestingly, the p53 point mutant mice had a similar phenotype to p53 family compound mutant mice suggesting that there is an interplay between the p53 family members in tumorigenesis and Li-Fraumeni syndrome.     

p63 Gene Mutations in EEC Syndrome, Limb-Mammary Syndrome, and Isolated Split Hand–Split Foot Malformation Suggest a Genotype-Phenotype Correlation

p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.     

Familial posterior fossa brain tumors of infancy secondary to germline mutation of the hSNF5 gene

We have identified a family afflicted over multiple generations with posterior fossa tumors of infancy, including central nervous system (CNS) malignant rhabdoid tumor (a subset of primitive neuroectodermal tumors, or PNET) and choroid plexus carcinoma. Various hereditary tumor syndromes, including Li-Fraumeni syndrome, Gorlin syndrome, and Turcot syndrome, have been linked to increased risk of developing CNS PNETs and choroid plexus tumors. Malignant rhabdoid tumors of the CNS and kidney show loss of heterozygosity at chromosome 22q11. The hSNF5 gene on chromosome 22q11 has recently been identified as a candidate tumor-suppressor gene in sporadic CNS and renal malignant rhabdoid tumors. We describe a family in which both affected and some unaffected family members were found to have a germline splice-site mutation of the hSNF5 gene, leading to exclusion of exon 7 from the mature cDNA and a subsequent frameshift. Tumor tissue shows loss of the wild-type hSNF5 allele, in keeping with a tumor-suppressor gene. These findings suggest that germline mutations in hSNF5 are associated with a novel autosomal dominant syndrome with incomplete penetrance that predisposes to malignant posterior fossa brain tumors in infancy.     

Is p53 intronic variant G13964C associated with predisposition to cancer?

Germline mutations of the p53 gene confer a high risk of diverse malignancies. The highest frequency of inherited p53 defects was noted in Li-Fraumeni syndrome (LFS), but almost half of the mutations were found in families with incomplete Li-Fraumeni-like syndrome (LFL), including familial breast cancer cases. Recently, a germline intronic G13964C base change of the p53 was reported as a high-risk mutation associated with familial breast cancer (LEHMAN et al. 2000). We genotyped Polish cancer patients and healthy control individuals for the G13964C variant. Patients were chosen from cancer families with phenotypes typical for germline mutations of p53 (LFS, LFL), BRCA1 [hereditary breast (ovarian) cancer, HB(O)C] or a complex consistent with both LFL and HB(O)C. Children with leukemia were included in the study as another high risk group (FELIX et al. 1992). The G13964C variant was detected in six of 87 (6.9%) cancer patients (including two ALL children), but also in eight of 96 (8.3%) control individuals (p > 0.4). Thus we found no evidence of the variant's association with a high risk of cancer.     

Mutations in C5ORF42 cause Joubert syndrome in the French Canadian population

Joubert syndrome (JBTS) is an autosomal-recessive disorder characterized by a distinctive mid-hindbrain malformation, developmental delay with hypotonia, ocular-motor apraxia, and breathing abnormalities. Although JBTS was first described more than 40 years ago in French Canadian siblings, the causal mutations have not yet been identified in this family nor in most French Canadian individuals subsequently described. We ascertained a cluster of 16 JBTS-affected individuals from 11 families living in the Lower St. Lawrence region. SNP genotyping excluded the presence of a common homozygous mutation that would explain the clustering of these individuals. Exome sequencing performed on 15 subjects showed that nine affected individuals from seven families (including the original JBTS family) carried rare compound-heterozygous mutations in C5ORF42. Two missense variants (c.4006C>T [p.Arg1336Trp] and c.4690G>A [p.Ala1564Thr]) and a splicing mutation (c.7400+1G>A), which causes exon skipping, were found in multiple subjects that were not known to be related, whereas three other truncating mutations (c.6407del [p.Pro2136Hisfs*31], c.4804C>T [p.Arg1602*], and c.7477C>T [p.Arg2493*]) were identified in single individuals. None of the unaffected first-degree relatives were compound heterozygous for these mutations. Moreover, none of the six putative mutations were detected among 477 French Canadian controls. Our data suggest that mutations in C5ORF42 explain a large portion of French Canadian individuals with JBTS.     

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.     

Joint effects of germ-line <Emphasis Type="Italic">TP53</Emphasis> mutation, <Emphasis Type="Italic">MDM2</Emphasis> SNP309, and gender on cancer risk in family studies of Li–Fraumeni syndrome

Li–Fraumeni syndrome (LFS) is a rare familial cancer syndrome characterized by early cancer onset, diverse tumor types, and multiple primary tumors. Germ-line TP53 mutations have been identified in most LFS families. A high-frequency single-nucleotide polymorphism, SNP309 (rs2279744), in MDM2 was recently confirmed to be a modifier of cancer risk in several case-series studies: substantially earlier cancer onset was observed in SNP309 G-allele carriers than in wild-type individuals by 7–16 years. However, cancer risk analyses that jointly account for measured hereditary TP53 mutations and MDM2 SNP309 have not been systematically investigated in familial cases. Here, we determined the combined effects of measured TP53 mutations, MDM2 SNP309, and gender and their interactions simultaneously in LFS families. We used the method that is designed for extended pedigrees and structured for age-specific risk models based on Cox proportional hazards regression. We analyzed the cancer incidence in 19 extended pedigrees with germ-line TP53 mutations ascertained through the clinical LFS phenotype. The dataset consisted of 463 individuals with 129 TP53 mutation carriers. Our analyses showed that the TP53 germ-line mutation and its interaction with gender were strongly associated with familial cancer incidence and that the association between MDM2 SNP309 and increased cancer risk was modest. In contrast with several case-series studies, the interaction between MDM2 SNP309 and TP53 mutation was not statistically significant in our LFS family cohort. Our results showed that SNP309 G-alleles were associated with accelerated tumor formation in both carriers and non-carriers of germ-line TP53 mutations.     

The Li-Fraumeni syndrome

Li-Fraumeni syndrome (LFS) has been the most common terminology used for the syndrome. It is a rare familial dominantly inherited cancer syndrome characterized by a wide spectrum of neoplasms occurring in children and young adults. The canonical definition of LFS includes a proband diagnosed with sarcoma before 45 years of age, a first-degree relative with cancer before this same age and another first- or second-degree relative in the lineage with any cancer before this age or sarcoma at any age. Multiple studies have reported p53 germline mutations in LFS families in various parts of the world. As in sporadic tumors, loss of heterozygosity leading to the inactivation of the wild-type allele by deletion or mutation is observed in LFS tumors. Cancer-risk in mutation carriers has been estimated to be 73% in males and nearly 100% in females, the difference almost entirely explained by breast cancer. The identification of germline p53 mutations in rare cancer-prone families has given rise to the medical, counseling, psychological and ethical problems.