Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC.

Approximately 130,000 cases of colorectal cancer (CRC) are diagnosed in the United States each year, and about 15% of these have a hereditary component. Two well-defined syndromes, familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC), account for up to 5% of the total new cases of CRC. Truncating APC mutations are responsible for FAP, and defective mismatch repair genes cause HNPCC. However, the genes responsible for most of the familial cases are unknown. Here we report a mutation (T to A at APC nucleotide 3920) found in 6% of Ashkenazi Jews and about 28% of Ashkenazim with a family history of CRC. Rather than altering the function of the encoded protein, this mutation creates a small hypermutable region of the gene, indirectly causing cancer predisposition.

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.

The accuracy of protein synthesis in reticulocyte and HeLa cell lysates.

The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [35S]cysteine with incorporation of [3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1-2) X 10(-3) cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 10(-6) cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.

A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-1.

The sensitivity and specificity of the polymerase chain reaction (PCR) for the detection of HIV-1 proviral DNA was determined in five laboratories with extensive experience in PCR testing. Five panels consisting of 105 HIV-1-seronegative specimens from regularly repeating blood donors with no risk factors for HIV infection and 99 HIV-1-seropositive and culture-positive specimens from a cohort of homosexual/bisexual men were sent under code to each laboratory. Amplification procedures and testing algorithms by which specimens were judged positive, negative, or indeterminate varied between laboratories. The average sensitivity for the five laboratories was 99.0%, with two laboratories achieving 100%. The average specificity was 94.7%, varying between 90.5 and 100%. The overall false-positive rate was 1.8%, the false-negative rate was 0.8%, and the indeterminate rate was 1.9%. Of 1,005 determinations made by the five laboratories, 32 (3.2%) were misclassifications. Most of the classification errors occurred in specimens from uninfected individuals and were distributed among the laboratories in such a way as to indicate laboratory error rather than the inherent reactivity of some samples. This emphasizes the need for standardization of PCR testing and caution in interpreting positive PCR reactions in HIV-1-seronegative persons.

Altered sensitivity of protein synthesis to paromomycin in extracts from aging human diploid fibroblasts.

Age-related differences in the effects of paromomycin (Pm) on protein synthesis have been investigated in translation reactions with extracts derived from young and old human diploid fibroblasts. Translation products from reactions directed by endogenous or exogenous mRNA were analyzed by polyacrylamide gel electrophoresis and fluorography. The exogenous mRNA lacked codons for cysteine, and therefore cysteine incorporation into translation products represented translational error. This laboratory has previously used this assay to show that the basal translational error level in the absence of Pm increases in extracts from old fibroblasts. In this report, Pm stimulated the misincorporation of cysteine by 6-7 fold over cysteine misincorporation levels in the absence of Pm. This degree of Pm stimulation was similar in extracts from young and old fibroblasts. However, other results showed quantitative differences in the responses to Pm between young and old cell extracts. Old cell extracts were less sensitive to the stimulation of the rate of protein synthesis, and more sensitive to the inhibition of protein synthesis, by Pm. It is proposed that aging human diploid fibroblasts contain altered ribosomes which react differently with Pm.

Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts.

The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks codons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 x 10(-5) cysteines/amino acid in young cell extracts to 2.9 x 10(-4) cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.

Metallothionein expression and stress responses in aging human diploid fibroblasts.

Metallothioneins (MTs) are low molecular weight proteins with a high cysteine content that are inducible by heavy metals and by other conditions of environmental stress. This laboratory was investigated in human diploid fibroblasts the induction of MTs by cadmium and by dexamethasone, and the induction of heat shock proteins, as models for age-related changes in gene expression that reflect the ability of old cells to respond to environmental stress. Old cells were more sensitive to the toxic effects of CdCl2 in the concentration range 100-175 microM. Analysis of 35S-cysteine-labelled cell extracts by polyacrylamide gel electrophoresis and fluorography showed that in the absence of any inducer, old cells have a 3.7-fold increase over young cells in the basal level of MT. The rate and extent of induction of MT by CdCl2 was reduced in old cells: Exposure of old cells to 100 microM CdCl2 for 18 h resulted in MT levels about 33% of the amount in young cells. Northern blot analysis showed that the changes in MT protein levels occurred in parallel with changes in mRNA levels, which implicates transcriptional control as the origin of these aging changes. These young/old differences in MT synthesis were maintained in density-arrested cultures, indicating that the aging changes were not due to differences in the cell cycle status of these cell populations. The rate and extent of induction of a 68-kDa heat shock protein were also reduced in old cells, which showed an increase in basal, uninduced level of this protein similar to MT. In contrast, old cells retained the ability to synthesize MTs in response to dexamethasone at a rate similar to that in young cells.

Café-au-lait spots and early onset colorectal neoplasia: a variant of HNPCC?

BACKGROUND: Café-au-lait spots (CALS) are classically found in neurocutaneous syndromes such as neurofibromatosis, but have not been associated with hereditary colorectal cancer. However, review of hereditary colorectal cancer case reports reveals occasional description of CALS on physical exam. METHODS: We describe the colonic and extracolonic phenotype in a family with CALS and early onset colorectal neoplasia (adenomas and/or cancer) and review 23 additional families reported in the literature. RESULTS: Among the 24 families, 32/59 (54.2%) individuals had colorectal adenomas diagnosed at a mean age of 15.7 +/- 1.1 (SE) years (range 5-38 years). The majority (24/32, 75.0%) of persons at first colorectal examination had oligopolyposis (< 100 polyps) versus polyposis (> or = 100 polyps). Forty-two of 59 (71.2%) individuals were affected with colorectal cancer, diagnosed at a mean age of 31.9 +/- 2.7 years (range 5-70 years). A brain tumor was found in 28/59 (47.5%) affected individuals (4 families with 2 or more cases) with an overall mean age of diagnosis of 16.5 +/- 1.2. Lymphoma and/or leukemia was found in 8/24 (33.3%) families (one family with 3 cases). Two families had mutation of the mismatch repair gene, hPMS2 (1 with homozygous germline mutation), while two carried homozygous germline mutations of another mismatch repair gene, hMLH1. CONCLUSIONS: Café-au-lait spots with early onset colorectal neoplasia may identify families with a variant of HNPCC characterized by oligopolyposis, glioblastoma at young age, and lymphoma. This variant may be caused by homozygous mutation of the mismatch repair genes, such as hPMS2 or hMLH1.

Human papillomavirus detection and p53 expression in clear-cell adenocarcinoma of the vagina and cervix.

OBJECTIVE: To determine whether infection with oncogenic human papillomavirus (HPV) and/or altered expression of the tumor suppressor protein p53 is associated with clear-cell adenocarcinoma of the vagina or cervix. METHODS: Paraffin-embedded tissue specimens were studied from 14 women with clear-cell adenocarcinoma of the vagina or cervix. Nine women had a history of intrauterine diethylstilbestrol exposure. Human papillomavirus DNA was amplified via the polymerase chain reaction using consensus L1 primers and was detected by dot blot hybridization with a generic HPV probe and type-specific oligonucleotide probes. P53 protein was detected by immunohistochemical analysis with a mouse monoclonal antibody, DO-7. RESULTS: Three tumors contained HPV 31 DNA sequences. Eight tumors were HPV DNA-negative and three were indeterminate for HPV. P53 was detected in ten tumors; it was undetectable in the three tumors containing HPV 31 and in one tumor indeterminate for HPV. Three patients presented with or later developed metastatic disease. In each case, the tumor, including sites of metastasis, was HPV-negative and p53-positive. CONCLUSIONS: These findings suggest that infections with oncogenic HPVs may be a cofactor in the development of clear-cell adenocarcinoma of the vagina or cervix, though this association is less than that reported for squamous or non-clear-cell adenocarcinomas. Prior studies have shown that detection of the p53 protein by immunohistochemistry correlates with mutations in the p53 tumor suppressor gene. The detection of p53 in HPV-negative clear-cell adenocarcinoma suggests a second mechanism in the etiology of these rare tumors.

Reduction in heat shock gene expression correlates with increased thermosensitivity in senescent human fibroblasts.

The expression of three major classes of heat shock genes was examined in human diploid cells at differing in vitro ages. Metabolic labeling of cellular proteins following a brief heat shock showed that the synthesis of heat shock proteins was significantly reduced in late-passage cells. Northern blot analyses revealed that the reduced expression of heat shock proteins in old cells correlated with a reduced accumulation of heat shock-specific transcripts. The attenuation of heat shock gene activity in senescent cells was not unique to thermal stress since exposure of cells to sodium arsenite (10-50 microM) elicited a similar response. The reduced expression of heat shock gene products correlated with an increased thermal lability in late-passage cells following acute hyperthermic (49 degrees C) exposure. The preinduction of heat shock genes protected cells against the lethal effects of acute hyperthermia and abolished the increased thermal lability observed in senescent cells. The reduced expression of the heat shock response demonstrates that old cells possess a diminished ability to withstand adverse environmental conditions and maintain homeostasis.

p53 protein expression and gene analysis in clear cell adenocarcinoma of the vagina and cervix.

OBJECTIVE: p53 is the most commonly mutated gene in human cancers. The objective of this study was to determine if clear cell adenocarcinomas (CCAs) of the vagina and cervix are associated with p53 gene mutations or alterations in p53 tumor-suppressor protein expression. METHODS: Paraffin-embedded tissue specimens from 21 women (median age 22 years) with clear cell adenocarcinoma of the vagina or cervix were studied. Fifteen women had a prior history of in utero exposure to diethylstilbestrol. p53 protein expression was detected by immunohistochemical (IHC) analysis with monoclonal antibody DO-7 (Dako Corp.) which recognizes both wild-type and mutant p53 proteins. For p53 gene analysis, genomic DNA from malignant tissue was isolated and exons 4-10 were amplified by PCR and subjected to mutation screening by single-stranded conformation polymorphism (SSCP) analysis. RESULTS: p53 protein was detected by IHC in tumors from 14 of 21 cases (67%). The observed p53 staining patterns were heterogeneous in both the proportion and intensity of tumor cells stained but were clearly overexpressed relative to the surrounding benign stroma. Metastatic tumors from 3 women with metastatic disease were also positive for p53 staining. SSCP analysis did not identify p53 mutations in any of the cases and strongly suggests that the tumors contained only wild-type p53 alleles. CONCLUSIONS: Recent studies have demonstrated that wild-type p53 may accumulate in response to DNA damage which normally leads to growth arrest or programmed cell death. Our observations are consistent with the hypothesis that p53 overexpression in CCAs of the vagina and cervix is a response to generalized DNA damage, rather than a result of p53 protein half-life prolongation resulting from mutational inactivation of p53. Overexpression of wild-type p53 protein in vaginal and cervical CCA may relate to the more favorable prognosis of this subset of tumors in comparison to other gynecologic tumors containing mutated p53 genes.

Human breast tumors containing non-DNA-binding immunoreactive (67 kDa) estrogen receptor.

Evidence to date indicates that structurally abnormal estrogen receptor (variant ER) can be detected in some human breast tumors. Based on in vitro ability to bind DNA sequences containing the cognate estrogen response element (ERE), these variant receptors may be categorized into DNA-binding ER (Type-1 variants) and non-DNA-binding ER (Type-2 variants). To look for Type-2 variants of normal size (67 kDa ER) that lack the ability to form immunoreactive ER-ERE complexes, a panel of 40 cryopreserved primary breast tumors were extracted and analyzed by enzyme immunoassay (ER-EIA), gel-shift, and Western blot techniques. For the 33 tumor extracts containing > or = 10 fmol/mg ER (by ER-EIA), the amount of 67 kDa ER detectable by D75 anti-ER monoclonal antibody under fully denatured and reduced assay conditions (Western blotting) did not correlate well with the presence or intensity of D75 immunoreactive ER-ERE bands seen under native conditions by gel-shift assay. Overall, 30% (10 of 33) of these extracts containing 67 kDa ER failed to produce immunoreactive ER-ERE complexes, with this frequency varying from over 40% in tumor samples with lower ER content (10-49 fmol/mg) to 11% in tumor samples with the highest ER content (> 100 fmol/mg). These results indicate that Type-2 variant receptors characterized as non-DNA-binding 67 kDa ER may be present in a significant fraction of ER-positive primary breast tumors; preliminary evidence suggests that further study of abnormalities in ER tertiary or quaternary structure, such as those produced by intracellular oxidation of ER thiol groups, is warranted.

Identification of DNA mismatch repair gene mutations in hereditary nonpolyposis colon cancer patients.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant disease caused by germline mutations in DNA mismatch repair genes. The mutational spectrum in these genes appears to be diverse, in both the distribution and the nature of the mutations. However, most described mutations generate a premature stop codon and ultimately result in the synthesis of a truncated protein. We have employed an in vitro transcription/translation assay to identify germline mutations in DNA mismatch repair genes from patients suspected of belonging to HNPCC kindreds. Our results suggest that this approach will be highly effective in identifying mutations in these patients and may lead to a reliable diagnostic test for the pre-symptomatic identification of HNPCC.

Hepatoblastoma and APC gene mutation in familial adenomatous polyposis.

BACKGROUND: Hepatoblastoma is a rare, rapidly progressive, usually fatal childhood malignancy, which if confined to the liver can be cured by radical surgical resection. An association between hepatoblastoma and familial adenomatous polyposis (FAP), which is due to germline mutation of the APC (adenomatous polyposis coli) gene, has been confirmed, but correlation with site of APC mutation has not been studied. AIM: To analyse the APC mutational spectrum in FAP families with hepatoblastoma as a possible basis to select kindreds for surveillance. PATIENTS: Eight patients with hepatoblastoma in seven FAP kindreds were compared with 97 families with identified APC gene mutation in a large Registry. METHODS: APC gene mutation was evaluated by RNase protection assay or in vitro synthesis protein assay. The chi 2 test and correlation were used for data analysis. RESULTS: APC gene mutation was identified in all seven FAP kindreds in which an at risk member developed hepatoblastoma. A male predominance was noted (six of eight), similar to literature cases (18 of 25, p < 0.01. Mutations were restricted to codons 141 to 1230, but no significant difference in site of mutation between pedigrees with and without hepatoblastoma was identified. CONCLUSIONS: Hepatoblastoma occurs primarily in boys in FAP kindreds and is associated with germline APC mutation in the 5' end of the gene. However, the site of APC mutation cannot be used to predict occurrence of this extracolonic cancer in FAP pedigrees.

HLA-DQ alpha allele and genotype frequencies in various human populations, determined by using enzymatic amplification and oligonucleotide probes.

Allele and genotype frequencies at the HLA-DQ alpha locus have been determined by the use of polymerase chain reaction (PCR) amplification and nonradioactive oligonucleotide probes. The probes define six alleles and 21 genotypes in a dot-blot format. A total of over 1,400 individuals from 11 populations has been typed by two different laboratories using this method. In contrast to some variable-number-of-tandem-repeat markers that have been used for identity determination, DQ alpha genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium in all populations studied. The distribution of alleles varies significantly between most of these populations. In Caucasians, the allele frequencies range from 4.3% to 28.5%. In this population, the power of discrimination is .94, and, for paternity determination, the power of exclusion is .642. These population data will allow the use of the HLA-DQ alpha marker in paternity determination, the analysis of individual identity in forensic samples, and anthropological studies.

Attenuated familial adenomatous polyposis (AFAP). A phenotypically and genotypically distinctive variant of FAP.

BACKGROUND: The usual manifestation of familial adenomatous polyposis (FAP) is hundreds or thousands of colonic adenomas. The authors previously described a colon cancer-prone syndrome characterized by fewer adenomas (1-100), most located in the proximal colon, and upper gastrointestinal lesions, particularly fundic gland polyps and duodenal adenomas. The colonic adenomas are often flat rather than polypoid, a feature emphasized in earlier reports with the term "hereditary flat adenoma syndrome." The syndrome has an autosomal dominant pattern of inheritance and is linked to the adenomatous polyposis coli (APC) locus at 5q. METHODS: This is a descriptive study based on one family that was followed for more than a decade. Total cell RNA was isolated from cultured lymphoblasts, and an in vitro protein synthesis assay was used to detect APC mutations. Sixteen individuals whose APC mutation status was known had sequential endoscopic evaluations. Five patients were given one or more courses of sulindac. RESULTS: There was perfect concordance between clinical affected status and an APC mutation. All affected members generated a 16-kDa polypeptide from the mutant allele, consistent with a 2-base pair deletion at the extreme 5' end of the APC gene. Sixteen mutation-positive individuals underwent upper gastrointestinal endoscopy and colonoscopy; 13 had colonic adenomas, with the number visualized at any one examination ranging from 1 to greater than 50. Upper gastrointestinal examination revealed fundic gland polyps in 15, gastric or duodenal adenomas in 4, and periampullary carcinoma in 1. CONCLUSION: AFAP is a phenotypically distinctive syndrome, differing from classic FAP by having fewer colonic adenomas that tend to be proximally distributed and flat rather than polypoid. The position of the APC germline mutation appears to allow for the molecular differentiation between FAP and the attenuated variant in that the extreme 5' APC mutations are associated with the latter.

Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.

Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.

The use and interpretation of commercial APC gene testing for familial adenomatous polyposis.

BACKGROUND: The use of commercially available tests for genes linked to familial cancer has aroused concern about the impact of these tests on patients. Familial adenomatous polyposis is an autosomal dominant disease caused by a germ-line mutation of the adenomatous polyposis coli (APC) gene that causes colorectal cancer if prophylactic colectomy is not performed. We evaluated the clinical use of commercial APC gene testing. METHODS: We assessed indications for APC gene testing, whether informed consent was obtained and genetic counseling was offered before testing, and the interpretation of the results through telephone interviews with physicians and genetic counselors in a nationwide sample of 177 patients from 125 families who underwent testing during 1995. RESULTS: Of the 177 patients tested, 83.0 percent had clinical features of familial adenomatous polyposis or were at risk for the disease-both valid indications for being tested. The appropriate strategy for presymptomatic testing was used in 79.4 percent (50 of 63 patients). Only 18.6 percent (33 of 177) received genetic counseling before the test, and only 16.9 percent (28 of 166) provided written informed consent. In 31.6 percent of the cases the physicians misinterpreted the test results. Among the patients with unconventional indications for testing, the rate of positive results was only 2.3 percent (1 of 44). CONCLUSIONS: Patients who underwent genetic tests for familial adenomatous polyposis often received inadequate counseling and would have been given incorrectly interpreted results. Physicians should be prepared to offer genetic counseling if they order genetic tests.