Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

Detailed computational study of p53 and p16: using evolutionary sequence analysis and disease-associated mutations to predict the functional consequences of allelic variants.

Deciding whether a missense allelic variant affects protein function is important in many contexts. We previously demonstrated that a detailed analysis of p53 intragenic conservation correlates with somatic mutation hotspots. Here we refine these evolutionary studies and expand them to the p16/Ink4a gene. We calculated that in order for 'absolute conservation' of a codon across multiple species to achieve P<0.05, the evolutionary substitution database must contain at least 3(M) variants, where M equals the number of codons in the gene. Codons in p53 were divided into high (73% of codons), intermediate (29% of codons), and low (0 codons) likelihood of being mutation hotspots. From a database of 263 somatic missense p16 mutations, we identified only four codons that are mutational hotspots at P<0.05 (8 mutations). However, data on function, structure, and disease association support the conclusion that 11 other codons with > or =5 somatic mutations also likely indicate functionally critical residues, even though P0.05. We calculated p16 evolution using amino acid substitution matrices and nucleotide substitution distances. We looked for evolutionary parameters at each codon that would predict whether missense mutations were disease associated or disrupted function. The current p16 evolutionary substitution database is too small to determine whether observations of 'absolute conservation' are statistically significant. Increasing the number of sequences from three to seven significantly improved the predictive value of evolutionary computations. The sensitivity and specificity for conservation scores in predicting disease association of p16 codons is 70-80%. Despite the small p16 sequence database, our calculations of high conservation correctly predicted loss of cell cycle arrest function in 75% of tested codons, and low conservation correctly predicted wild-type function in 80-90% of codons. These data validate our hypothesis that detailed evolutionary analyses help predict the consequences of missense amino-acid variants.

TP53 mutations in breast cancer associated with BRCA1 or BRCA2 germ-line mutations: distinctive spectrum and structural distribution.

Several groups have studied the molecular pathology of inherited breast cancer. By combining several such studies, we show in this study that somatic TP53 abnormalities are more common in breast cancer associated with BRCA1 or BRCA2 germ-line mutations than in sporadic breast cancers (odds ratio, 2.8; P = 0.0003). Then, we compared the spectrum of TP53 mutations for breast cancers in the IARC TP53 mutation database with the 82 mutations reported in BRCA1/2-associated breast cancers. The spectrum differed significantly both in distribution (P < 1 x 10(-6)) and in base changes (P = 0.025). Mutations at A:T bp were more common in BRCA1/2-associated tumors and strand bias suggesting DNA repair abnormalities was found. Changes were common at TP53 codons that are not mutation hotspots. Structural modeling showed that most of these p53 non-hotspot amino acids characterized in breast tumors isolated from patients with deficient BRCA1/2 function are distributed in a region of the protein on the opposite side of the p53 DNA-binding surface. Our results suggest that BRCA1/2 mutations influence the type and distribution of TP53 mutations seen in breast cancer.

Genotype-specific Trp53 mutational analysis in ultraviolet B radiation-induced skin cancers in Xpc and Xpc Trp53 mutant mice.

We have examined the mutational spectrum in the Trp53 gene from UVB radiation-induced skin cancers in Trp53+/+ and Trp53+/- mutant mice of all three possible Xpc genotypes. Mutations were detected in exons 2-10 of the Trp53 coding region in approximately 90% of >80 different skin cancers examined. In contrast to Trp53+/+ mice in which most mutations in the Trp53 gene were located in exons 5-8, the majority of the mutations in Trp53+/- mice were at other exons. We observed a high predilection for C-->T transition mutations at a unique CpG site in codon 122 (exon 4) of the Trp53 gene in Xpc-/- Trp53+/- mice. This site is not part of a pyrimidine dinucleotide. Mutations at this codon, as well as in codons 124 and 210, were observed exclusively in Xpc-/- or Xpc+/- mice. Mutations at the corresponding codons (127 and 213) in the human p53 gene have been reported in skin tumors from human patients with xeroderma pigmentosum. Hence, mutations at codons 122 (125), 124 (127), and 210 (213) may constitute signatures for defective or deficient nucleotide excision repair in mice (humans). In Xpc-/- mice, the majority of mutations were located at C residues in CpG sites, in which the C is presumably methylated. A similar bias can be deduced from studies in human XP individuals.

Defective nucleotide excision repair in xpc mutant mice and its association with cancer predisposition.

Mice that are genetically engineered are becoming increasingly more powerful tools for understanding the molecular pathology of many human hereditary diseases, especially those that confer an increased predisposition to cancer. We have generated mouse strains defective in the Xpc gene, which is required for nucleotide excision repair (NER) of DNA. Homozygous mutant mice are highly prone to skin cancer following exposure to UVB radiation, and to liver and lung cancer following exposure to the chemical carcinogen acetylaminofluorene (AAF). Skin cancer predisposition is significantly augmented when mice are additionally defective in Trp53 (p53) gene function. We also present the results of studies with mice that are heterozygous mutant in the Apex (Hap1, Ref-1) gene required for base excision repair and with mice that are defective in the mismatch repair gene Msh2. Double and triple mutant mice mutated in multiple DNA repair genes have revealed several interesting overlapping roles of DNA repair pathways in the prevention of mutation and cancer.

Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features.

Missense mutations in p53 frequently occur at 'hotspot' amino acids which are highly conserved and represent regions of structural or functional importance. Using the p53 mutation database and the p53 DNA sequences for 11 species, we more precisely defined the relationships among conservation, mutation frequency and protein structure. We aligned the p53 sequences codon-by-codon and determined the degree of substitution among them. As a whole, p53 is evolving at an average rate for a mammalian protein-coding gene. As expected, the DNA binding domain is evolving more slowly than the carboxy and amino termini. A detailed map of evolutionary conservation shows that within the DNA binding domain there are repeating peaks and valleys of higher and lower evolutionary constraint. Mutation hotspots were identified by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Seventy-three hotspots were identified; these 19% of codons account for 88% of all reported p53 mutations. Both high evolutionary constraint and mutation hotspots are noted at amino acids close to the protein-DNA interface and at others more distant from DNA, often buried within the core of the folded protein but sometimes on its surface. The results indicate that targeting highly conserved regions for mutational and functional analysis may be efficient strategies for the study of cancer-related genes.

Does aflatoxin B1 play a role in the etiology of hepatocellular carcinoma in the United States?

Previous research showed that risk factors associated with hepatocellular carcinoma (HCC) include infection with hepatitis B (HBV) and hepatitis C (HCV) viruses, exposure to aflatoxin B1 (AFB1), and liver cirrhosis, due primarily to alcohol consumption. To determine whether AFB1 may play a role in HCC in the United States, a search for AFB1 adducts and p53 alterations, potentially induced by AFB1, was conducted in the United States in 23 HCC patients with available tissue samples. The presence of AFB1 tumor-DNA and -serum lysine adducts and mutant p53 product was determined by immunoassays and codon 249 p53 mutation by restriction enzyme analysis. HBV and HCV serology and serum HBV-DNA were also determined. Thirteen patients were positive for HBV by HBs antigen or anti-HBc antigen or by polymerase chain reaction for HBV-DNA sequences. Nine patients were free of HBV and HCV markers; 5 of 22 sera tested were anti-HCV positive. p53 Protein expression, determined by immunohistochemical staining, was present in 5 of the 23 tumor tissues, whereas p53 codon 249 mutations were not observed in the 5 cases in which tissue was available for study. AFB1 tumor-DNA adducts were present in 3 of 19 tumor tissues, and in 1 of these 3 samples p53 protein was also detected. Sera from only 5 of the patients were tested for AFB1-lysine adducts, and all were positive. In these five patients, neither p53 protein nor a mutation on codon 249 was detected. The demonstration that AFB1-DNA and -lysine adducts are present in HCC patients in the United States is intriguing but requires further substantiation because of the small number of subjects in this pilot study. To elucidate the pathogenetic significance of these findings, further investigation, including studies in larger patient cohorts and properly selected controls, is warranted.

Integrity of p53 in hepatitis B x antigen-positive and -negative hepatocellular carcinomas.

Inactivation of the tumor suppressor p53 seems to be important to the pathogenesis of hepatocellular carcinoma (HCC) associated with chronic hepatitis B virus infection. Although this inactivation may be due to mutations in the p53 gene, recent evidence suggests that the hepatitis B virus-encoded X antigen (HBxAg) binds to and inactivates wild-type p53. Hence, experiments were designed to test the hypothesis that there is a low frequency of p53 mutations in HBxAg-positive HCC. HBxAg and p53 were assayed by immunohistochemistry (IHC) in HCC and nontumor liver from 16 Chinese patients, half of whom were hepatitis B surface antigen carriers. HBxAg was detectable in tumor and/or nontumor cells from all patients by IHC; six of these samples also had detectable p53. To determine whether p53 detection by IHC, and hence stabilization, is associated with mutation, sequencing of p53 exons 5-8 was performed with each patient sample. Wild-type sequences were found in 13 of 16 HBxAg-positive cases (81%). Hence, HBxAg is a common marker of HCC that correlates with the persistence of wild-type p53 among both carriers and noncarriers. The low frequency of p53 mutations in HCC in these patients implies that p53 inactivation may occur predominantly by complex formation with HBxAg.

The Familial Cancer Program of the Vermont Cancer Center: Development of a Cancer Genetics Program in a Rural Area.

In response to many scientific discoveries linking cancer in certain families to inherited factors, the Vermont Cancer Center established the Familial Cancer Program (FCP) in December 1993. This multifaceted program combines the expertise of clinicians and researchers in many disciplines, including genetics, oncology, psychology, and molecular biology. The program's goals are identification of families in its region with excess cancer, provision of clinical services to such families, and use of research protocols when available and appropriate. This article describes the experience of setting up a familial cancer program in a rural area and discusses both successes and challenges in such an endeavor.

Deletions and insertions in the p53 tumor suppressor gene in human cancers: confirmation of the DNA polymerase slippage/misalignment model.

We analyzed all published deletions and insertions in the p53 gene to assess the relevance of mutagenesis models. Almost all deletions and insertions can be explained by one or more of the following DNA sequence features: monotonic base runs, adjacent or nonadjacent repeats of short tandem sequences, palindromes, and runs of purines or pyrimidines (homocopolymer runs). Increased length of monotonic runs correlates positively with increased frequency of events. Complex frameshift mutations can be explained by the formation of quasi-palindromes, with mismatch excision and replication using one strand of the palindrome as a template. Deletions and insertions in the p53 tumor suppressor gene may reflect both spontaneous and carcinogen-induced mutagenesis.

Phase I evaluation of therapy with four schedules of 5-fluorouracil by continuous infusion combined with recombinant interferon alpha.

The purpose of this study was to determine the maximum tolerated dose and dose-limiting toxicities of 5-fluorouracil (5-FU) when administered concurrently with recombinant IFN-alpha using four continuous infusion (CI) dosing schedules of 5-FU. Forty-five patients with advanced or refractory cancers were treated with 5-FU by CI, plus IFN during the infusion only, by one of four schedules: schedule A: 24-h 5-FU infusion repeated weekly, 9 x 10(6) units IFN x 2 doses weekly; schedule B: 48-h 5-FU infusion repeated weekly, 9 x 10(6) units IFN x 4 doses weekly; schedule C: 5-day 5-FU infusion repeated every 3 weeks, 9 x 10(6) units IFN three times weekly; and schedule D: 21-day 5-FU infusion, repeated after 7 days off therapy, 9 x 10(6) units IFN three times weekly. At least three patients were treated at all dose levels. Doses of 5-FU were escalated to the next level if less than one half of the patients at a given level developed grades 2-4 toxicity. The maximum tolerated dose for 5-FU was 2150 mg/m2/week for schedule A (24-h CI), 2350 mg/m2/week for schedule B (48-h CI), 750 mg/m2/day for schedule C (5-day CI), and 175 mg/m2/day for schedule D (21-day CI). Median delivered dose intensities at these levels were 1788 mg/m2/week for schedule A, 2192 mg/m2/week for schedule B, 1250 mg/m2/week for schedule C, and 593 mg/m2/week for schedule D. The dose-limiting toxicities were hematological and gastrointestinal (stomatitis, diarrhea, nausea, anorexia) for schedules A and B and gastrointestinal (mostly stomatitis) for schedules C and D. Severe fatigue due to IFN was rare. Responses correlated with toxicity >/= grade 2, but not with increased dose intensity. Responses were noted in several tumor types on schedules A, B, and D. 5-FU can be combined with IFN using 24- and 48-h high-dose and long-term low-dose CI schedules, with large differences in dose intensity at maximum tolerated dose. Shorter infusions produce less mucosal and more hematological toxicity. Tumor responses were seen on both short- and long-term CI schedules. Future studies can establish the efficacies of these new schedules of 5-FU/IFN administration in specific tumor types.

Molecular genetics of lung cancer.

The identification of genetic abnormalities in lung cancer and their consequences for cell growth, differentiation and function offer insights into normal cellular function and carcinogenesis. The integration of this knowledge with data from other fields, including epidemiology, toxicology, carcinogen metabolism, clinical genetics and clinical cancer therapy, promises advancements in lung cancer risk assessment, screening, prevention, prognostic assessment and therapy.

Database of p53 gene somatic mutations in human tumors and cell lines.

A data base is described in which over 2,500 mutations in the p53 gene of human tumors and tumor cell lines are compiled from a systematic search of reports published before 1 January 1994. Data from 1994 are being added intermittently, with a systematic search and update scheduled for December, 1994. The compilation has been deposited with the EMBL Data Library and is available in electronic form free of charge. This report contains a rationale for the compilation, a brief summary of the major findings and a description of the data base.

The type B receptor for tumor necrosis factor-alpha mediates DNA fragmentation in HL-60 and U937 cells and differentiation in HL-60 cells.

Tumor necrosis factor-alpha (TNF) binds to two specific cell surface receptors, types A and B, which are both present on HL-60 and U937 cells, and induces monocytoid differentiation in HL-60 cells and early DNA fragmentation in HL-60 and U937 cells. To further define the receptors' roles, we studied how monoclonal antibodies (MoAbs) against each receptor affected TNF-induced cellular responses. HTR-9, an MoAb against the type B (low affinity, 55 Kd) receptor, reproduced all of these effects in a dose-dependent manner. UTR-1, an MoAb against the type A (high affinity, 75 Kd) receptor, had no effect in saturating doses, but supersaturating doses enhanced DNA fragmentation threefold. TNF and interferon gamma (IFN-gamma) synergistically induced morphologic differentiation and monocytic antigen expression, while the antitype B receptor MoAb was synergistic for morphologic response, but not antigen expression. Our results indicate that (1) the type B receptor mediates some responses to TNF in HL-60 and U937 cells, (2) the type A receptor does not stimulate these responses, (3) the TNF molecule is not necessary for some of these actions, and (4) TNF-induced morphologic changes and surface antigen expression in HL-60 cells may be regulated by separate postreceptor pathways.