[Show abstract][Hide abstract] ABSTRACT: We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.
[Show abstract][Hide abstract] ABSTRACT: The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Apc mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Apc gene, Apc1638T, resulting in a truncated Apc protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc1638T/1638T animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.
Genes & Development 06/1999; 13(10):1309-21. DOI:10.1101/gad.13.10.1309 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of the genetic background on the tumor spectrum of Apc1638N, a mouse model for attenuated familial adenomatous polyposis (FAP), has been investigated in X-irradiated and untreated F1 hybrids between C57BL/6JIco-Apc1638N (B6) and A/JCrIBR (A/J), BALB/cByJIco (C) or C3H/HeOuJIco (C3). Similar to the ApcMin model, the Apc1638N intestinal tumor multiplicity seems to be modulated by Mom1. Moreover, several additional (X-ray-responsive) modifier loci appear also to affect the Apc1638N intestinal tumor number. The genetic background did not significantly influence the number of spontaneous desmoids and cutaneous cysts in Apc1638N. In general, X-irradiation increased the desmoid multiplicity in Apc1638N females but had no effect in males. The opposite was noted for the cyst multiplicity after X-rays. Surprisingly, X-irradiated CB6F1-Apc1638N females were highly susceptible to the development of ovarian tumors, which displayed clear loss of the wild-type Apc allele.
Genes Chromosomes and Cancer 04/1999; 24(3):191-8. · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer still represents one of the most common causes of morbidity and mortality among Western populations. The adenomatous polyposis coli (APC) gene, originally identified as the gene responsible for familial adenomatous polyposis (FAP), an inherited predisposition to multiple colorectal tumors, is now considered as the true "gatekeeper" of colonic epithelial proliferation. It is mutated in the vast majority of sporadic colorectal tumors, and inactivation of both APC alleles occurs at early stages of tumor development in man and mouse. The study of FAP has also led to one of the most consistent genotype-phenotype correlations in hereditary cancer. However, great phenotypic variability is still observed not only among carriers of the identical APC mutation from unrelated families but also from within the same kindred. The generation of several mouse models carrying specific Apc mutations on the same inbred genetic background has confirmed the genotype-phenotype correlations initially established among FAP patients, as well as provided important insights into the mechanisms of colorectal tumor formation. Here we review the major features of the available animal models for FAP and attempt the formulation of a hypothetical model for APC-driven tumorigenesis based on the observed genetic and phenotypic variability in mouse and man.
Cytogenetics and cell genetics 02/1999; 86(2):105-11. DOI:10.1159/000015361
[Show abstract][Hide abstract] ABSTRACT: Familial adenomatous polyposis (FAP) is characterised by hundreds of colorectal adenomas. Endocrine neoplasms have occasionally been reported, as have gastric polyps, which are usually hamartomatous in the fundus of the stomach and adenomatous in the antrum. A 57 year old man with colorectal, gastric, and periampullary adenomatous polyposis, in association with three bilateral adrenocortical adenomas, is presented. Mutation screening showed a 5960delA germline mutation in the adenomatous polyposis coli (APC) gene predicted to lead to a premature stop codon. This mutation was found in three of the four children of the patient. Western blot analysis of a lymphoblastoid cell line derived from the patient failed to detect any truncated APC polypeptide. This rare 3' mutation is responsible for an unusually complex and late onset phenotype of FAP.
Journal of Medical Genetics 02/1999; 36(1):65-7. DOI:10.1136/jmg.36.1.65 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Germ-line mutations in DNA mismatch-repair genes (MSH2, MLH1, PMS1, PMS2, and MSH6) cause susceptibility to hereditary nonpolyposis colorectal cancer. We assessed the prevalence of MSH2 and MLH1 mutations in families suspected of having hereditary nonpolyposis colorectal cancer and evaluated whether clinical findings can predict the outcome of genetic testing.
We used denaturing gradient gel electrophoresis to identify MSH2 and MLH1 mutations in 184 kindreds with familial clustering of colorectal cancer or other cancers associated with hereditary nonpolyposis colorectal cancer. Information on the site of cancer, the age at diagnosis, and the number of affected family members was obtained from all families.
Mutations of MSH2 or MLH1 were found in 47 of the 184 kindreds (26 percent). Clinical factors associated with these mutations were early age at diagnosis of colorectal cancer, the occurrence in the kindred of endometrial cancer or tumors of the small intestine, a higher number of family members with colorectal or endometrial cancer, the presence of multiple colorectal cancers or both colorectal and endometrial cancers in a single family member, and fulfillment of the Amsterdam criteria for the diagnosis of hereditary nonpolyposis colorectal cancer (at least three family members in two or more successive generations must have colorectal cancer, one of whom is a first-degree relative of the other two; cancer must be diagnosed before the age of 50 in at least one family member; and familial adenomatous polyposis must be ruled out). Multivariate analysis showed that a younger age at diagnosis of colorectal cancer, fulfillment of the Amsterdam criteria, and the presence of endometrial cancer in the kindred were independent predictors of germ-line mutations of MSH2 or MLH1. These results were used to devise a logistic model for estimating the likelihood of a mutation in MSH2 and MLH1.
Assessment of clinical findings can improve the rate of detection of mutations of DNA mismatch-repair genes in families suspected of having hereditary nonpolyposis colorectal cancer.
New England Journal of Medicine 09/1998; 339(8):511-8. DOI:10.1056/NEJM199808203390804 · 55.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been estimated that the prevalence of carriers of a mutated mismatch repair (MMR) gene among the general population in Western countries is between 5 and 50 per 10,000. These carriers have a risk of >85% of developing colorectal carcinoma (CRC) and therefore need careful follow-up. The objective of this study was to analyze the cost-effectiveness of CRC surveillance of carriers of a mutated MMR gene.
The authors constructed a model to estimate the potential health effects (life expectancy) and healthcare costs of two strategies: 1) surveillance, with colonoscopy every 2-3 years, and 2) no CRC surveillance. Estimates of the lifetime risk of developing CRC and the stage distribution of CRC for symptomatic patients were derived from the Dutch hereditary nonpolyposis colorectal carcinoma (HNPCC) registry. The CRC stage specific relative survival rates and the effectiveness of surveillance in preventing or detecting cancer early were based on Finnish studies. The costs of surveillance and treatment were derived from recent American studies.
The results showed that 1) surveillance of gene carriers led to an increase in life expectancy of 7 years, and 2) the costs of surveillance under a wide range of assumptions are less than the costs of no CRC surveillance.
CRC surveillance of HNPCC gene carriers appears to be effective and considerably less costly than no CRC surveillance and therefore deserves to be supported by governmental agencies and health insurance organizations.
Cancer 05/1998; 82(9):1632-7. DOI:10.1002/(SICI)1097-0142(19980501)82:9<1632::AID-CNCR6>3.3.CO;2-X · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: About 15% of patients with colorectal cancer report a family history of this disease. An estimated 1%-5% of patients have hereditary non-polyposis colorectal cancer (HNPCC). Recently, DNA mismatch repair genes associated with this syndrome were identified. For about 50% of families in which HNPCC occurs, DNA-based diagnosis and presymptomatic DNA testing are now feasible. Diagnosis of a hereditary tumour syndrome is relevant for both the patient with cancer and his or her close relatives. The complexities of family studies warrant the forming of a multidisciplinary team which may choose to work within a specialized cancer family clinic.
Recent results in cancer research. Fortschritte der Krebsforschung. Progrès dans les recherches sur le cancer 02/1998; 146:20-31.
[Show abstract][Hide abstract] ABSTRACT: Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to the formation of multiple colorectal adenomas. Moreover, patients with FAP are at high risk of developing several extracolonic manifestations, including desmoids, cutaneous cysts, and tumors of the upper gastrointestinal tract. Although by definition desmoids are nonmalignant, because of their aggressive invasion of local structures, they represent one of the major causes of morbidity and mortality among patients with FAP.
This study describes the histopathologic and molecular characterization of Apc1638N, a mouse model for the broad spectrum of extracolonic manifestations characteristic of FAP.
Heterozygous Apc+/Apc1638N animals develop fully penetrant and multifocal cutaneous follicular cysts and desmoid tumors in addition to attenuated polyposis of the upper gastrointestinal tract. Moreover, breeding of Apc+/Apc1638N mice in a p53-deficient background results in a dramatic seven-fold increase of the desmoid multiplicity.
Because of the attenuated nature of their intestinal phenotype, these mice survive longer than other murine models for Apc-driven tumorigenesis. Therefore, Apc1638N represents an ideal laboratory tool to test various therapeutic intervention strategies for the management of intestinal as well as extraintestinal tumors.
[Show abstract][Hide abstract] ABSTRACT: Seven-week-old Apc1638N mice were exposed to a single dose of 5 Gy total-body X-irradiation resulting in a 8-fold increase in the number of intestinal tumors and a reduction of the lifespan to an average of 6 months. The distribution of tumors along the intestinal tract as well as the adenoma/carcinoma ratio, were similar between non-irradiated and irradiated animals. Semi-quantitative PCR analysis of intestinal-tumor DNA revealed that 10 out of 14 tumors had lost the wild-type Apc allele. However, in contrast to spontaneous Apc1638N intestinal tumors in which the LOH event at the Apc locus involves the entire chromosome 18 (1), in 6 out of 10 tumors derived from X-irradiated animals the Apc loss is associated with only a partial intrachromosomal deletion. The remaining tumors have lost all chromosome 18 markers tested. In addition to the intestinal tumors, female Apc1638N mice are susceptible to the development of mammary tumors. Upon X-irradiation, Apc1638N mice show a striking 15-fold increase in mammary tumors. Moreover, Apc1638N mice spontaneously develop other extra-intestinal neoplasia, such as desmoid-like lesions similar to those associated with familial adenomatous polyposis (FAP), the human syndrome caused by germline mutations in the APC gene. Spontaneous desmoid growth is sex-dependent, as male Apc1638N mice develop 3-fold more desmoids than female mice. Interestingly, X-irradiation seemed to increase the number of desmoids per animal nearly twofold only in female Apc1638N mice. Five out of 9 desmoids found in Apc1638N mice exposed to X-ray displayed loss of the wild-type Apc allele.
[Show abstract][Hide abstract] ABSTRACT: Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.
The American Journal of Human Genetics 09/1997; 61(2):329-35. DOI:10.1086/514847 · 10.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an effort to generate a good mouse model for human colorectal cancer, we generated mice which carry a mutation in the adenomatous polyposis coli (Apc) gene. Mice which are heterozygous for the mutation, designated Apc1638, develop colonic polyps and tumors of the small intestine. Neoplasms were found in 96% of animals studied, and they included adenomas, adenocarcinomas, and polypoid hyperplasias. The mice developed an average of 3.3 tumors, with the highest number in duodenum, followed by jejunum, stomach, ileum, and colon. Focal areas of dysplasias were observed in the colonic mucosa in 50% of mice which were 10 months old or older. These results suggest that mice carrying the Apc1638 mutation can serve as a good model to study the initiation, progression, and inhibition of gastrointestinal tumors.
Journal of Experimental Zoology 02/1997; 277(3):245-54. DOI:10.1002/(SICI)1097-010X(19970215)277:3<245::AID-JEZ5>3.0.CO;2-O
[Show abstract][Hide abstract] ABSTRACT: Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. We screened the entire coding region of the APC gene for mutations in an unselected series of 105 Dutch FAP kindreds. For the analysis of exons 1-14, we employed the GC-clamped denaturing gradient gel electrophoresis (DGGE), while the large exon 15 was examined using the protein truncation test. Using this approach, we identified 65 pathogenic mutations in the above 105 apparently unrelated FAP families. The mutations were predominantly either frameshifts (39/65) or single base substitutions (18/65), resulting in premature stop codons. Mutations that would predict abnormal RNA splicing were identified in seven cases. In one of the families, a nonconservative amino acid change was found to segregate with the disease. In spite of the large number of APC mutations reported to date, we identified 27 novel germline mutations in our patients, which reiterates the great heterogeneity of the mutation spectrum in FAP. In addition to the point mutations identified in our patients, structural rearrangements of APC were found in two pedigrees, by Southern blot analysis. The present study indicates that the combined use of DGGE, protein truncation test, and Southern blot analysis offers an efficient strategy for the presymptomatic diagnosis of FAP by direct mutation detection. We found that the combined use of the currently available molecular approaches still fails to identify the underlying genetic defect in a significant subset of the FAP families. The possible causes for this limitation are discussed.
Human Mutation 02/1997; 9(1):7-16. DOI:10.1002/(SICI)1098-1004(1997)9:1<7::AID-HUMU2>3.3.CO;2-7 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three germline mutations in the TP53 tumor-suppressor gene are reported, two of which are not reported previously. A missense mutation at codon 265 of TP53 was found in three patients of a family that complied with the definition of the Li-Fraumeni syndrome. A nonsense mutation in codon 306 was found in a woman who had had a rhabdomyosarcoma at age 4 and a subsequent breast cancer at age 22. She was part of a Li-Fraumeni-like family, but the parental origin of the mutation could not be traced. Finally, while screening for somatic alterations in TP53 in a series of 141 sporadic breast tumors, we detected a constitutional missense mutation in codon 235 in a woman diagnosed with breast cancer at age 26 and a recurrence 4 years later. The recurrence, but not the primary tumor, showed an additional missense mutation at codon 245 as well as loss of the wild-type allele. This suggests that the 245 mutation was particularly important for tumor progression and that there might exist heterogeneity in terms of cancer predisposition potential among the various germline TP53 mutations.
Human Mutation 01/1997; 9(2):157-63. DOI:10.1002/(SICI)1098-1004(1997)9:2<157::AID-HUMU8>3.0.CO;2-6 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps predominantly in the colorectal region. Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for most cases of FAP. Mutations at the 5' end of APC are known to be associated with a relatively mild form of the disease, called attenuated adenomatous polyposis coli (AAPC). We identified a frameshift mutation in the 3' part of exon 15, resulting in a stop codon at 1862, in a large Dutch kindred with AAPC. Western blot analysis of lymphoblastoid cell lines derived from affected family members from this kindred, as well as from a previously reported Swiss family carrying a frameshift mutation at codon 1987 and displaying a similar attenuated phenotype, showed only the wild-type APC protein. Our study indicates that chain-terminating mutations located in the 3' part of APC do not result in detectable truncated polypeptides and we hypothesize that this is likely to be the basis for the observed AAPC phenotype.
Human Genetics 01/1997; 98(6):727-34. DOI:10.1007/s004390050293 · 4.82 Impact Factor