Cancer Research Grants Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heinimann, K.
Right arrow Articles by Dobbie, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heinimann, K.
Right arrow Articles by Dobbie, Z.
[Cancer Research 61, 7616-7622, October 15, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

Nontruncating APC Germ-line Mutations and Mismatch Repair Deficiency Play a Minor Role in APC Mutation-negative Polyposis1

Karl Heinimann2,,3, Annick Thompson4, Andreas Locher4, Tamara Furlanetto4, Eva Bader, Angela Wolf, Remy Meier, Klaus Walter, Peter Bauerfeind, Giancarlo Marra, Hansjakob Müller, Dorothee Foernzler and Zuzana Dobbie

Research Group Human Genetics, Division of Medical Genetics, University Clinics, 4031 Basel, Switzerland [K. H., A. L., T. F., E. B., A. W., H. M., Z. D.]; F. Hoffmann-La Roche, 4070 Basel, Switzerland [A. T., D. F.]; Department of Gastroenterology, Kantonsspital, 4410 Liestal, Switzerland [A. W., R. M.]; Department of Surgery, Kreiskrankenhaus, 79618 Rheinfelden, Germany [K. W.]; Department of Gastroenterology, University Clinics, 8091 Zurich, Switzerland [P. B.]; and Institute for Medical Radiobiology, University of Zurich, 8008 Zurich, Switzerland [G. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Familial adenomatous polyposis, an autosomal-dominantly inherited colorectal cancer predisposition syndrome, is caused by germ-line mutations in the adenomatous polyposis coli (APC) gene. Despite the use of different screening methods, studies worldwide fail to identify APC mutations in 20–50% of all familial adenomatous polyposis patients (APC mutation-negatives). In this study, missense mutations in the coding region of the APC gene, which would have been missed by the protein truncation test, as well as mutations in the APC promoter and the 3' untranslated region, were determined by the single nucleotide polymorphism discovery assay and direct DNA sequencing in 31 mutation-negative polyposis patients. Seventeen gene alterations were identified, whereof four (12.9%) represent possibly pathogenic germ-line mutations: silent A290T (promoter) and A8822G (3' untranslated region) as well as missense R99W and E1317Q (coding region). The 27 remaining, truly APC mutation-negative polyposis patients displayed a significantly later age at diagnosis compared with APC mutation carriers (46.1 versus 35.2 years; P < 0.01). APC mutation-negative individuals with >100 colonic polyps were more likely to present with extracolonic disease (P < 0.05) than those with <100. Assessment of microsatellite instability (MSI), a hallmark of mismatch repair deficiency, in 68 tumors from 21 truly APC mutation-negative patients, identified 4 (5.9%) unstable tubulo-villous adenomas (3 MSI-High and 1 MSI-Low), stemming from 4 (19%) unrelated individuals and likely to be caused by hMLH1 promoter hypermethylation. In conclusion, only a small proportion of APC germ-line mutation carriers is missed by the protein truncation test, and mismatch repair deficiency does not seem to substantially contribute to tumor development in APC mutation-negative polyposis patients.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FAP5 (MIM 175100) is an autosomal dominantly inherited CRC predisposition syndrome. During adolescence, colonic polyposis ensues in virtually all FAP patients with the development of a few dozen to several thousands of adenomatous polyps throughout the colorectum, some of which eventually progress to CRC during the fourth and fifth decade if left untreated (1) . According to the Danish Polyposis Register, the mean annual incidence amounts to 1.85 x 10-6 (time period, 1971–1992), with both sexes equally affected; FAP patients were found to account for approximately 0.1% of all Danish CRC patients (2) .

FAP is caused by germ-line mutations within the APC gene, which was first localized to chromosome 5q21–22 in 1986 and finally cloned in 1991 (3, 4, 5, 6, 7) . Applying different screening methods, like PTT and single strand conformation polymorphism analysis, more than 400 different germ-line mutations have been described, most of which lead to a truncated APC protein (8 , 9) .6 Despite these screening efforts, 20–50% of all classical FAP patients remain without a detectable APC germ-line mutation (so-called APC mutation-negative; Refs. 8 , 10, 11, 12, 13, 14, 15 ).

In addition, the disease displays considerable phenotypic heterogeneity. Even within so-called "typical" FAP families, polyp number and age of onset may vary considerably (reviewed in Ref. 16 ). The attenuated form of FAP (AAPC) has been characterized by a low (<100) and highly heterogeneous number of colonic polyps and later age of disease onset (17, 18, 19) . AAPC is mainly observed with mutations located at the 5' or the 3' end of the APC gene, but may also be associated with missense variants, such as I1307K and E1317Q (20 , 21) .

In comparison with FAP patients with an identified mutation, APC mutation-negative polyposis patients display later age at diagnosis (45.3 versus 35.2 years) and 50% have <100 colorectal polyps (sometimes also referred to as multiple adenoma patients; Refs. 22 , 23 ). Additionally, they tend to have fewer extracolonic manifestations (e.g., polyps of the upper gastrointestinal tract, desmoids, and osteomas). An extremely mild disease phenotype combined with a positive family history may mimic the clinical characteristics of another CRC predisposition, HNPCC. Intriguingly, recent observations suggest that some of these APC mutation-negative FAP families, unlinked to the APC locus, are linked to a new gene, CRAC1, on chromosome 15q (24) . Other candidate genes for APC mutation-negative patients may include the MMR loci and the TGFBIIR locus (25) .

To elucidate the molecular events in APC mutation-negative polyposis patients, the present study aimed to: (a) identify missense mutations in the coding region of the APC gene missed by the standard screening method (PTT); (b) assess the presence of mutations in the APC promoter and the 3' UTR region; (c) delineate the phenotypic characteristics of the truly APC mutation-negative polyposis patients; and (d) determine the extent to which MSI, a hallmark of MMR deficiency, contributes to the pathogenesis of APC mutation-negative polyposis.


   SUBJECTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
From March 1996 to August 2000, 100 unrelated Caucasian patients were referred by their treating physicians to the Department of Medical Genetics, University of Basel, Basel, Switzerland, for APC gene mutation analysis because of clinical diagnosis of polyposis coli. Written informed consent was obtained from all individuals.

In 31 (28%) polyposis coli patients, no truncating APC mutation in the germ-line could be detected (so-called APC mutation-negatives) using PTT, which has been described previously (26) . Having no family history suggestive of HNPCC, all APC mutation-negative patients were analyzed additionally for the presence of germ-line mutations in the coding (GenBank accession no. M73548), the promoter (U02509), and the 3' UTR region (AC008416) of the APC gene by applying two complementary methods, the SNP discovery assay (27) and DNA cycle sequencing. To assess the frequency of a given gene alteration in the population, a control panel of 146 healthy Caucasians was used, with 100 individuals stemming from the Coriell Cell Repositories (HD100CAU; Coriell Institute of Medical Research, Camden, NJ) and 46 healthy probands from the Medical Genetics Department, University Clinics, Basel, Switzerland.

DNA Extraction from Peripheral Blood.
Genomic DNA was isolated from EDTA blood according to the method described previously (28) . Briefly, 10 ml of blood were mixed with 30 ml of EL buffer [155 mM NH4 Cl, 10 mM KHCO3, and 1 mM EDTA (pH 7.4)] and left on ice for 15 min. The lysate was centrifuged at 2000 rpm for 10 min, washed twice with EL buffer, and the intact leukocyte pellet resuspended in NL buffer [10 mM Tris-HCl (pH 8.2), 400 mM NaCl, 2 mM Na2 EDTA, 1% SDS, and 200 µg/ml proteinase K] and incubated overnight at 37°C. The next day, 1 ml of 6 M NaCl was added, it was vigorously shaken, and then it was centrifuged to remove cellular proteins. The supernatant containing the DNA was placed in a fresh tube, and the DNA was precipitated with ethanol. The resulting DNA pellet was washed with 70% ethanol, dried briefly, and then suspended in 1 ml of TE buffer [10 mM Tris-HCl (pH 7.5) and 0.1 M EDTA].

SNP Discovery Assay.
Forty ng of genomic DNA were PCR-amplified in 50 µl of reactions using an MJ Research Tetrad PCR machine. To amplify the promoter region, coding sequence, and 3' UTR of the APC gene, primer pairs which amplify 300–600-bp fragments were designed either manually or using Primer3 software7 and GenBank sequence information (accession no. M73548). Standard reaction conditions included the following reagents: 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 pmol of each primer, and 1.5 units Taq Polymerase (Boehringer Mannheim, Mannheim, Germany). For some primer pairs the Invitrogen PCR buffer optimization kit was used for the adaptation of magnesium concentration and pH. For the amplification of GC-rich fragments, either 10% DMSO was added, or the amplification was done with solution Q from the Qiagen Hot Start Polymerase Kit. Qiagen Hot Start Taq Polymerase was used for a stringent PCR amplification.

The thermocycling protocol consisted of an initial incubation of 95°C for 15 min and then 35 cycles of 94°C for 1 min, 60°C for 30 s, 72°C for 1 min, and one final extension step of 72°C for 10 min. After PCR amplification, fragments were purified with the Qiaquick PCR purification kit on a Biorobot 9600. Cycle sequencing was performed on an MJ Research Tetrad PCR machine using ABI Big Dye Terminator chemistry according to the manufacturer’s instructions, with the following changes: 2–4 µl of purified PCR product was mixed with 2 µl of Big Dye Terminator Ready-Reaction-Premix and 10 pmol of sequencing primer; 5% DMSO for GC-rich sequences. The final reaction volume was 10 µl. Reactions were subjected to 28 cycles at 93°C for 30 s, 48°C for 30 s, and 58°C for 2 min and then an ethanol precipitation. After decanting the ethanol, samples were evaporated to dryness using a SpeedVac for 2 min and resuspended in 45 µl of ultrapure water. An ABI 3700 capillary sequencer was loaded with 2.5 µl using POP5 as a polymer.

SNP Analysis.
The sequencer-generated data were transferred to a Sun server, where the analysis software, a suite of sequence assembly tools (Phred, Phrap, Polyphred, and Consed), resides (29, 30, 31) . Phred is the base caller, which also assigns a quality score to each position in the read (29 , 30) . Phrap assembles the reads into a contig. Consed is the analysis viewer (31) , a graphical user interface to view or edit the assembly created by Phrap. Polyphred has an algorithm to detect heterozygote polymorphisms in an assembly (32) . Because reads from PCR products may, in some cases, continue beyond the physical end of the DNA, the low quality sections of the reads were removed from the end of the read files before Phrap and Polyphred analysis. Phrap and Polyphred generated "tags," which were used to indicate the position and nature of different sequence features. Whereas Phrap-generated tags essentially deal with features relevant to the assembly of the contig, Polyphred-generated tags indicate the position and type of observed polymorphisms. These results were verified manually.

Cycle Sequencing.
Cycle sequencing was performed using the Thermo Sequenase cycle sequencing kit according to the manufacturer’s instructions (Amersham Pharmacia, Dübendorf, Switzerland). The primers were end-labeled with an IR dye, IRD-800 (MWG Biotech, Ebersberg, Germany), and used directly in the cycle-sequencing reaction. Briefly, 2–5 µl of PCR product were diluted to a final volume of 7 µl. Ten units of exonuclease I and 2 units of shrimp alkaline phosphatase (both Amersham Pharmacia) were added to remove excess primers and dNTPs (at 37°C for 15 min and 80°C for 15 min). To each sample, dH20 was added to obtain a final volume of 25 µl from which 6 µl were combined with 2 µl of A, C, G, or T reagent, respectively. Cycle sequencing conditions were 95°C for 4 min, as an initial denaturing step, and then 25 cycles at 95° for 30 s, 55° for 30 s, and 72° for 1 min. Finally, 3 µl of red stop dye were added to accomplish the reaction. After denaturation at 95°C for 5 min, 1 µl of the reaction mix was loaded on a 6% denaturing polyacrylamide gel and analyzed on an automated DNA sequencer according to the manufacturer’s instructions (LI-COR, Lincoln, NE).

Tumor DNA Extraction.
After histopathological classification of H&E-stained tissue slides, a representative portion of the tumor (adenoma or carcinoma) with an average tumor content of >=70% was scraped off, and DNA extraction was performed according to the QIAamp tissue kit protocol (Qiagen, Basel, Switzerland). Briefly, 180 µl of buffer ATL and 20 µl proteinase K (20 ng/µl) were added to each tumor sample, which then were incubated overnight at 55°C for digestion until the tissue was completely lysed. Subsequently, 200 µl of buffer AL were added and incubated at 70°C for 10 min, with the subsequent addition of 210 µl of ethanol (100%), and mixed thoroughly by vortexing. Applied on a QIAamp spin column, the lysate was centrifuged at 10,000 rpm for 3 min. After having discarded the filtrate, 500 µl of washing buffer AW were added twice and centrifuged at full speed (14,000 rpm). Finally, the DNA was eluted twice with 200 µl of buffer AE.

Microsatellite Marker Analysis.
Ten microsatellite markers were chosen based on recommendations of the National Cancer Institute workshop on MSI. The first panel included the microsatellite loci BAT25, BAT26, D5S346, D17S250, and D2S123, which were analyzed using a commercially available kit (Roche Diagnostics, Basel, Switzerland). The second panel of microsatellites included BAT40, MYCL1, D10S197, D18S58, and D18S69, which were optimized for multiplex PCR. One hundred ng of leukocyte DNA and 200 ng of tumor DNA were used to perform the PCR under the following conditions: first panel: initial denaturation at 94°C for 2 min and then 30 cycles at 94°C for 10 s, 55°C for 30 s, and 72°C for 30 s with a final extension step at 72°C for 7 min; second panel: initial denaturation at 94°C for 3 min and then 30 cycles of 94°C for 20 s, 56°C for 30 s, and 72°C for 45 s, and a final extension step at 72°C for 5 min. Two µl of PCR product were added to 10 µl formamide (including 0.5 µl GS size standard 350 Tamra), denatured at 95°C for 5 min, chilled on ice, and loaded on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Rotkrenz, Switzerland). The interpretation of the presence of MSI, defined as the occurrence of novel alleles, followed the recommendations of the National Cancer Institute workshop: none of the microsatellite markers unstable, stable (MSS); >0–<=30% of markers unstable, low instability (MSI-L); and >30% of markers unstable, high instability (MSI-H). Tumor samples from HNPCC patients were included as positive controls. Microsatellite marker analysis also allowed the identification of LOH, commonly defined as a >50% reduction in relative intensity of one allele compared with the other (33) .

Immunohistochemical Analysis.
Four-µm serial sections from paraffin blocks were mounted on silanized slides, deparaffinized, and rehydratated. Antigen retrieval was obtained by heating the sections in a pressure cooker at 120°C for 2 min in 10 mM citrated-buffered solution (pH 6.0). DAKO peroxidase blocking reagent and goat serum were sequentially used to suppress nonspecific staining caused by endogenous peroxidase activity and nonspecific binding of antibodies, respectively. Incubations with primary monoclonal antibodies were performed as follows: anti-hMSH2, 24 h at 4°C with Ab NA26 (Oncogene Research), 1 µg/ml; anti-hMSH6, 2 h at room temperature with Ab G70220 (Transduction Laboratories), 4 µg/ml; anti-hMLH1, 1 h at room temperature with Ab 13271A (PharMingen), 1.2 µg/ml; and anti-hPMS2, 24 h at 4°C with Ab 65861A (PharMingen), 3 µg/ml. After washing, antimouse secondary antibodies conjugated to peroxidase-labeled polymer (DAKO EnVision+kit) were applied for 30 min at room temperature, and the peroxidase activity was developed by incubation with 3,3'diaminobenzidine chromogen solution (DAKO). Sections were then counterstained slightly with hematoxylin.

Despite the use of several commercially available antibodies against APC, no reproducible and reliable immunohistological staining patterns could be established to directly assess the loss of APC protein expression in the tumors available.

Phenotypical Characteristics and Statistical Analysis.
The APC mutation-negative polyposis patients were compared using phenotypical data (gender, age at diagnosis, polyp number, CRC occurrence, and extracolonic disease) and molecular genetic data (mutational analysis, MSI, and LOH). The term "multiple," noted in the medical records of some patients, describes adenoma numbers between >5 and <100 adenomas (34) . For statistical analysis, the {chi}2, Fisher’s exact, and Student t tests were used when appropriate, with all probabilities reported as 2-tailed Ps, considering a P <= 0.05 to be significant (Excel 2000; Microsoft Corp.).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
From a consecutive series of 100 patients clinically diagnosed as having polyposis coli between March 1996 and August 2000, 31 (31%) were found to carry no detectable truncating APC mutation as assessed by PTT. These so-called APC mutation-negative polyposis patients were consecutively screened for germ-line mutations within the coding region of the APC gene as well as the promoter and the 3' UTR. Furthermore, interindividual phenotypic differences and the presence of MSI in colonic tumors (mostly adenomatous polyps) from the truly APC mutation-negative patients were assessed. Because of the unavailability of additional family members and/or the uninformativeness of the markers used, linkage to the APC locus could not be established in APC mutation-negative patients with a positive family history.

Mutational Analysis of the Promoter, Coding and 3' UTRs of the APC Gene.
Applying the SNP discovery assay and direct DNA sequencing on 31 APC mutation-negative polyposis patients, 17 germ-line variants were detected within the APC promoter, coding, and 3' UTR (Table 1)Citation . Thirteen variants were deemed to be nonpathogenic, because they did not lead to an amino acid change and thus did not have an obvious consequence on the protein level. Although possible effects on transcription and splicing efficiency cannot be excluded (35) , these polymorphisms were also present in healthy control persons and did not cosegregate with the disease phenotype within the family (Table 1)Citation . Therefore, they have been considered as nonpathogenic polymorphisms, and the patients carrying them have been considered as truly APC mutation-negative.


View this table:
[in this window]
[in a new window]

  		Table 1 Point mutations identified in a consecutive series of apparently APC mutation-negative polyposis coli patients (n = 31)

 
In four (12.9%) patients, four different, possibly pathogenic, mutations were identified: whereas the missense mutations R99W and E1317Q have been reported previously (20 , 36) , the A290T mutation, located within the APC promoter, as well as the A8822G mutation in the 3' UTR of the APC gene represent novel mutations and are, to our knowledge, the first mutations described within regulatory regions of APC.

The R99W missense mutation (C358T) was identified in a 64-year-old woman (1506) with a positive family history of CRC and diagnosed of having <100 adenomas, predominantly located in the ascending colon and of tubular origin, and having no extracolonic disease at presentation. The missense mutation was identified also in her daughter, who did not develop polyps until 38 years of age.

Male patient 1762, harboring the E1317Q mutation (G4012C), without a family history of polyposis and/or CRC, was diagnosed as having about 90 mostly tubular and tubulo-villous adenomas in the large intestine as well as moderately differentiated adenocarcinoma of the rectum at 44 years of age. No extracolonic disease was reported.

In the family of the male patient 1551, who was diagnosed as having <100 colorectal adenomas at 47 years of age, the A290T germ-line mutation was found to cosegregate with disease (i.e., polyposis and/or CRC) in two affected family members, whereas an unaffected family member (age 41) was found to carry only wild-type alleles of the gene. The A290T promoter mutation was absent in a control population consisting of 146 healthy Caucasian individuals.

The 3' UTR A8822G germ-line mutation was identified in a male patient (1736), who was diagnosed at 46 years of age as having classical colonic polyposis (>100 adenomas) as well as 2 synchronous adenocarcinomas of the caecum and descending colon, respectively. Because of the negative family history, no cosegregation analysis could be performed. In the Caucasian control population, however, the A8822G mutation was found in only one (0.7%) individual.

Subsequently, the four individuals carrying possibly pathogenic APC germ-line mutations were excluded from the additional phenotypic analysis of truly APC mutation-negative polyposis patients.

Phenotypic Properties of Truly APC Mutation-negative Patients.
The phenotypic properties of the 27 APC mutation-negative polyposis patients are depicted in Table 2Citation . The sex ratio (male:female) was 16:11 (59%:41%) and did not significantly covary with other phenotypic characteristics (i.e., age, family history, polyp number, presence of CRC and extracolonic disease). The polyposis was clinically diagnosed at a mean age of 46.1 years (±14.7, SD; range, 20–73), which is significantly later compared with our APC-positive patients’ mean age (35.2 years ± 13.7, SD; P < 0.01). Although not statistically significant (P = 0.20), women were found to be diagnosed on average 7.8 years earlier than men (41.5 ± 17.2 years versus 49.3 ± 12.3 years, respectively). In addition, there were no significant differences in age distribution when grouped according to family history, polyp number, occurrence of CRC, and extracolonic manifestations. Subdividing the APC mutation-negative polyposis patients according to the polyp number observed at diagnosis (<100/multiple versus >100 adenomas) demonstrated that patients exhibiting >100 adenomas were more likely to present with extracolonic disease (Fisher’s exact test; P < 0.05). They did not, however, differ with respect to any other phenotypic characteristics investigated; in particular, a similar percentage of patients with a positive and those with a negative family history displayed >100 polyps at diagnosis (25 and 27%, respectively). Extracolonic disease tended to be more often observed in patients with a positive family history (71%; n = 7) compared with those without (35%; n = 20), although this was not statistically significant (P = 0.1).


View this table:
[in this window]
[in a new window]

  		Table 2 Phenotypic properties of 27 APC mutation-negative polyposis patients

 
MSI in Tumors from APC Mutation-negative Polyposis Patients.
From 21 (78%) of 27 APC mutation-negative individuals, paraffin-embedded adenoma and/or adenocarcinoma tissue specimens were available for MSI testing. Importantly, the 21 patients displayed nearly identical phenotypic properties (i.e., gender, age at diagnosis, family history, polyp number, CRC and extracolonic disease) when compared with the overall group (data not shown). In total, 77 tissue specimens encompassing 74 colorectal adenomas and 3 adenocarcinomas were analyzed, with MSI status established in 68 (88%) samples. In addition, tumor tissue (n = 13) from all four patients (1506, 1551, 1736, and 1762) harboring possibly pathogenic APC germ-line mutations as well as adenocarcinomas from two hMLH1 mutation carriers (proven MSI-H) were investigated as controls.

The results of the MSI analysis for each of the 21 APC mutation-negative polyposis patients are summarized in Table 3Citation . Four (5.9%) of 68 tumors were found to exhibit a low (MSI-L) or high (MSI-H) degree of instability, almost exclusively in dinucleotide repeats. Histologically, they were characterized as moderately differentiated, tubular, or tubulo-villous adenomas, stemming from four (19%) unrelated individuals. Significantly, all unstable adenomas belonged to individuals having >100 colonic adenomas at diagnosis (P < 0.01), with two patients also having a positive family history. All adenomas (n = 13) coming from the four patients with possibly pathogenic APC germ-line mutations were found to be stable (MSS), and the MSI-H status of positive controls could be confirmed.


View this table:
[in this window]
[in a new window]

  		Table 3 Summary of MSI analysis results in 21 APC mutation-negative polyposis patients

 
From patient 1465, two tubulo-villous adenomas from the descending and the sigmoid colon could be investigated, with one being MSI-H [four of eight markers (50%)] and one MSS for all markers investigated. Unfortunately, immunohistochemical analysis of the unstable adenoma was inconclusive, because the biopsy specimen was too small and did not include normal mucosal epithelium for adequate comparison. The woman was 64 years of age when colonic polyposis (>100 adenomas) was discovered, with no apparent CRC or extracolonic disease present. In two family members (her son and her mother) polyposis coli had been diagnosed at 31 and 73 years of age, respectively, with the latter individual having also developed cancer of the ascending colon.

From patient 1505, two tubulo-villous adenomas from the distal colon as well as a rectal adenocarcinoma were available for study, with one adenoma displaying MSI-H [three of eight markers (38%)] and the remaining two tissues being MSS. Immunohistochemical analysis (for hMLH1, hMSH2, hMSH6, and hPMS2) of the unstable adenoma revealed a very weak hMLH1 staining pattern in the adenoma portion when compared with adjacent normal mucosa. The woman was diagnosed with polyposis (>100 adenomas) at 45 years of age, when rectal cancer had been discovered also. Her family history was negative for polyposis and/or CRC, and no extracolonic disease was present.

For patient 1777, a total of nine tumor specimens were investigated, encompassing four tubular and four tubulo-villous adenomas as well as an adenocarcinoma of the rectum. One tubular adenoma was found to be MSI-H [three of eight markers (38%)]. Immunohistochemically, all four MMR genes investigated displayed similar staining patterns in the adenoma and the adjacent normal mucosa. The female patient was diagnosed with polyposis (>100) at 22 years of age, without a family history of polyposis/CRC or extracolonic disease. Twenty-two years after subtotal colectomy, she developed a rectal adenocarcinoma at 44 years of age.

For patient 1552, one of four tubulo-villous adenomas investigated was found to display a low degree instability at marker D18S58 [one of eight markers (13%)]. In contrast to hMSH2, hMSH6, and hPMS2 proteins, being equally present in adenoma and normal mucosa, the hMLH1 protein staining pattern was very weak in the adenoma portion. The man had a negative family history of CRC and was diagnosed with polyposis coli (>100 adenomas) at 72 years of age, with no CRC present. At 76 years of age, he developed an abdominal desmoid tumor at the site where the coloectomy had been performed 4 years before.

LOH was observed in various adenomas from different APC mutation-negative individuals, predominantly involving the dinucleotide repeat markers D18S58 and D18S69, both located on chromosome 18q21 close to the tumor suppressor loci DCC and SMAD4, as well as MYCL1, a tetranucleotide repeat, located upstream of L-myc on chromosome 1p34 (Table 3)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, 31 APC mutation-negative patients, stemming from a consecutive series of 100 polyposis coli patients and carrying no truncating APC germ-line mutation as determined by PTT, underwent extensive APC mutational screening using the SNP discovery assay and direct DNA sequencing. Four (13%) possibly pathogenic germ-line mutations were identified in the coding sequence (R99W and E1317Q) as well as in the promoter (A290T) and the 3' UTR (A8822G) of the gene.

In agreement with previous reports on genotype/phenotype correlations at the 5' end of the APC gene, the patient harboring the R99W mutation displayed a markedly milder phenotype (i.e., <100 polyps, later age at diagnosis, absence of extracolonic disease; Refs. 17 , 37 ). Although a matter of ongoing debate, the E1317Q missense mutation is believed either to directly exert a (mildly?) pathogenic effect by a yet-undetermined mechanism or to hallmark a cryptic APC mutation nearby, predisposing to the development of multiple colorectal adenomas and carcinoma with a variable degree of penetrance (20) . In agreement with previous reports, the patient displayed <100 polyps at the time of diagnosis of rectal cancer at 44 years of age (20) .

The A290T promoter mutation was found to cosegregate with disease and was absent in a control series of 146 Caucasian controls, which clearly argues in favor of the mutation being pathogenic. Thus far, no firm conclusions can be made concerning the pathogenicity of the A8822G germ-line alteration in the 3' UTR of the APC gene, because cosegregation analysis was not possible, and 1 of 148 healthy Caucasian controls displayed the same mutation. Clearly, systematic screening of the 3' UTR region in other APC mutation-negative collectives as well as investigations on the functional relevance of the A8822G mutation are needed to determine its putatively pathogenic role. Moreover, use of the diploid-to-haploid gene conversion method should help to evaluate the significance of the mutations within the regulatory regions and definitely exclude the presence of masked APC mutations (38) .

Albeit with limitations (i.e., failure to detect large genomic deletions/insertions), the results of the current extensive mutation analysis indicate that only a small proportion (4 of 31; 12.9%) of APC mutation carriers is missed by standard truncation detection techniques such as PTT, and that in a considerable percentage of polyposis patients (27 of 100; 27%) other, yet-undetermined, pathogenetic mechanisms may play a role. In particular, previous work failed to identify pathogenic germ-line mutations in another member of the Wnt signaling pathway, the ß-catenin gene, in 6 of 27 APC mutation-negative patients of the current survey (39) . It remains to be seen, however, to which degree mutations in other members of the Wnt pathway, such as axin1 and axin2, contribute to the disease.

The age at diagnosis of the remaining 27 truly APC mutation-negative polyposis patients significantly differed from mutation-positive individuals, in that the former were diagnosed 10 years later on average (46.1 versus 35.2 years; P < 0.01). However, despite the significant difference in age at diagnosis, both groups were diagnosed with CRC 20–30 years (fourth and fifth decades) earlier than the general Swiss population (seventh decade; Swiss Cancer Registries’ Association database, 1996), pointing toward an inherited genetic disorder rather than a sporadic event. Overall, the phenotypic properties such as age at diagnosis, number of polyps, polyp site, and histology, as well as development of CRC and extracolonic disease, did not significantly differ among the APC mutation-negative polyposis patients, with one exception: patients exhibiting >100 adenomas presented more frequently with extracolonic disease than patients with multiple/<100 adenomas (P < 0.05). Finally, as our study population represents a highly selected group, i.e., APC mutation-negative polyposis patients, an ascertainment bias cannot be ruled out.

To determine the degree to which a deficient MMR system might contribute to the pathogenesis in APC mutation-negative patients, MSI analysis was assessed in 68 adenomas from 21 patients, identifying MSI in 4 (5.9%) adenomas (3 MSI-H and 1 MSI-L) from 4 (19%) different patients. Significantly, all four patients had developed >100 adenomas at diagnosis. The proportion of unstable adenomas was comparable with other reports on MSI in FAP and sporadic polyps (3%; Ref. 40 ). Immunohistochemically, hMLH1 staining was very weak in two of three adenomas suitable for analysis, both stemming from patients with a negative family history. As MSI did not occur in all tumors analyzed from the same patient, these findings point to the somatic inactivation of hMLH1 by promoter hypermethylation as frequently observed in unstable sporadic adenomas and CRCs, rather than to the presence of an hMLH1 germ-line alteration (41 , 42) . Taken together, MSI in adenomas from APC mutation-negative polyposis patients seems to be a rare event, comparable with frequencies observed in sporadic ones, and mainly attributable to hMLH1 inactivation, most likely by promoter hypermethylation. Thus, MMR deficiency is unlikely to account for tumor development in APC mutation-negative polyposis patients, which stands in agreement with previous reports assessing the frequency of MMR gene mutations in APC mutation-negative multiple adenoma patients (43) . Moreover, a study on familial CRC patients without polyposis coli and reminiscent of HNPCC found no pathogenic germ-line alterations in the 5' AAPC region (i.e., exons 1–6) of the APC gene, indicating that mutations in this region are infrequent in familial nonpolyposis CRC patients (44) .

In conclusion, our findings indicate that only a small proportion (13%) of APC germ-line mutation carriers is missed by conventional screening techniques (i.e., PTT), and that in 30% of polyposis patients no alterations within the APC gene can be identified. These APC mutation-negative polyposis patients are characterized by a significantly later age at diagnosis, compared with mutation carriers, and patients with >100 polyps are more frequently found to present with extracolonic disease. As MSI was present in only a fraction of adenomas from APC mutation-negative polyposis patients, a significant contribution of MMR deficiency to the tumorigenesis in these patients seems unlikely. Future research on the molecular genetic events and pathways involved in tumor development in APC mutation-negative polyposis patients may not only help to improve genetic counseling and medical care for these patients and their families, but may also shed additional light on the genetic basis of CRC in general.


   ACKNOWLEDGMENTS
 
We thank all of the patients and families who participated in this study, their respective doctors and pathologists for contributing clinical information and tumor material, Ritva Haider and Michèle Attenhofer-Häner for excellent technical assistance, and Dr. Ian Tomlinson for critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the Swiss Cancer League KFS 932-09-1999, Swiss National Science Foundation Grant 200-049310.96, Uarda Frutiger-Fonds of the Freiwillige Akademische Gesellschaft Basel, Jubilaeumsstiftung of the Schweizerische Lebens- und Rentenanstalt für Volksgesundheit und medizinische Forschung, and the Emilia Guggenheim-Schnurr Foundation, Basel, Switzerland. Back

2 Present address: Molecular Population and Genetics Laboratory, Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom. Back

3 To whom requests for reprints should be addressed, at Research Group Human Genetics, University Clinics, Hebelstrasse 20, 4031 Basel, Switzerland. Phone: 4161-265-2362; Fax: 4161-265-2350; E-mail: karl.heinimann{at}unibas.ch Back

4 These authors contributed equally to the work. Back

5 Abbreviations: FAP, familial adenomatous polyposis; CRC, colorectal cancer; APC, adenomatous polyposis coli; AAPC, attenuated form of FAP; MMR, mismatch repair; UTR, untranslated region; MSI, microsatellite instability; MSI-H, high instability; MSI-L, low instability; HNPCC, hereditary nonpolyposis colorectal carcinoma; PTT, protein truncation test; SNP, single nucleotide polymorphism; LOH, loss of heterozygosity; MSS, microsatellite stable. Back

6 See also the universal APC mutation database. Internet address: http://www.umd.necker.fr:2008. Back

7 Rozen, S., and Skaletsky, H. J., Primer3 (1998). Code available at Internet address: http://iwww.genome.wi.mit.edu/genome-software/other/primer3.html. Back

Received 5/ 7/01. Accepted 8/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bufill J. A. Colorectal cancer genetics. Closing the gap between genotype and phenotype. Cancer (Phila.), 76: 2389-2392, 1995.[Medline]
  2. Bulow S., Faurschou Nielsen T., Bulow C., Bisgaard M. L., Karlsen L., Moesgaard F. The incidence rate of familial adenomatous polyposis. Results from the Danish Polyposis Register. Int. J. Colorectal Dis., 11: 88-91, 1996.[Medline]
  3. Herrera L., Kakati S., Gibas L., Pietrzak E., Sandberg A. A. Gardner syndrome in a man with an interstitial deletion of 5q. Am. J. Med. Genet., 25: 473-476, 1986.[Medline]
  4. Bodmer W. F., Bailey C. J., Bodmer J., Bussey H. J., Ellis A., Gorman P., Lucibello F. C., Murday V. A., Rider S. H., Scambler P., et al Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature (Lond.), 328: 614-616, 1987.[Medline]
  5. Leppert M., Dobbs M., Scambler P., O’Connell P., Nakamura Y., Stauffer D., Woodward S., Burt R., Hughes J., Gardner E., et al The gene for familial polyposis coli maps to the long arm of chromosome 5. Science (Wash. DC), 238: 1411-1413, 1987.[Abstract/Free Full Text]
  6. Kinzler K. W., Nilbert M. C., Su L. K., Vogelstein B., Bryan T. M., Levy D. B., Smith K. J., Preisinger A. C., Hedge P., McKechnie D., et al Identification of FAP locus genes from chromosome 5q21. Science (Wash. DC), 253: 661-665, 1991.[Abstract/Free Full Text]
  7. Groden J., Thliveris A., Samowitz W., Carlson M., Gelbert L., Albertsen H., Joslyn G., Stevens J., Spirio L., Robertson M., et al Identification and characterization of the familial adenomatous polyposis coli gene. Cell, 66: 589-600, 1991.[Medline]
  8. Nagase H., Nakamura Y. Mutations of the APC (adenomatous polyposis coli) gene. Hum. Mutat., 2: 425-434, 1993.[Medline]
  9. Laurent-Puig P., Beroud C., Soussi T. APC gene: database of germline and somatic mutations in human tumors and cell lines. Nucleic Acids Res., 26: 269-270, 1998.[Abstract/Free Full Text]
  10. Miyoshi Y., Ando H., Nagase H., Nishisho I., Horii A., Miki Y., Mori T., Utsunomiya J., Baba S., Petersen G., et al Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients. Proc. Natl. Acad. Sci. USA, 89: 4452-4456, 1992.[Abstract/Free Full Text]
  11. Groden J., Gelbert L., Thliveris A., Nelson L., Robertson M., Joslyn G., Samowitz W., Spirio L., Carlson M., Burt R., et al Mutational analysis of patients with adenomatous polyposis: identical inactivating mutations in unrelated individuals. Am. J. Hum. Genet., 52: 263-272, 1993.[Medline]
  12. Armstrong J. G., Davies D. R., Guy S. P., Frayling I. M., Evans D. G. APC mutations in familial adenomatous polyposis families in the Northwest of England. Hum. Mutat., 10: 376-380, 1997.[Medline]
  13. Giardiello F. M., Petersen G. M., Piantadosi S., Gruber S. B., Traboulsi E. I., Offerhaus G. J., Muro K., Krush A. J., Booker S. V., Luce M. C., Laken S. J., Kinzler K. W., Vogelstein B., Hamilton S. R. APC gene mutations and extraintestinal phenotype of familial adenomatous polyposis. Gut, 40: 521-525, 1997.[Abstract/Free Full Text]
  14. van der Luijt R. B., Khan P. M., Vasen H. F., Tops C. M., van Leeuwen-Cornelisse I. S., Wijnen J. T., van der Klift H. M., Plug R. J., Griffioen G., Fodde R. Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis. Hum. Mutat., 9: 7-16, 1997.[Medline]
  15. Scott R. J., Meldrum C., Crooks R., Spigelman A. D., Kirk J., Tucker K., Koorey D. Familial adenomatous polyposis: more evidence for disease diversity and genetic heterogeneity. Gut, 48: 508-514, 2001.[Abstract/Free Full Text]
  16. Sieber O. M., Tomlinson I. P., Lamlum H. The adenomatous polyposis coli (APC) tumour suppressor—genetics, function and disease. Mol. Med. Today, 6: 462-469, 2000.[Medline]
  17. Spirio L., Olschwang S., Groden J., Robertson M., Samowitz W., Joslyn G., Gelbert L., Thliveris A., Carlson M., Otterud B., et al Alleles of the APC gene: an attenuated form of familial polyposis. Cell, 75: 951-957, 1993.[Medline]
  18. Friedl W., Meuschel S., Caspari R., Lamberti C., Krieger S., Sengteller M., Propping P. Attenuated familial adenomatous polyposis due to a mutation in the 3' part of the APC gene. A clue for understanding the function of the APC protein. Hum. Genet., 97: 579-584, 1996.[Medline]
  19. van der Luijt R. B., Meera Khan P., Vasen H. F., Breukel C., Tops C. M., Scott R. J., Fodde R. Germline mutations in the 3' part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli. Hum. Genet., 98: 727-734, 1996.[Medline]
  20. Frayling I. M., Beck N. E., Ilyas M., Dove-Edwin I., Goodman P., Pack K., Bell J. A., Williams C. B., Hodgson S. V., Thomas H. J., Talbot I. C., Bodmer W. F., Tomlinson I. P. The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history. Proc. Natl. Acad. Sci., 95: 10722-10727, 1998.[Abstract/Free Full Text]
  21. Laken S. J., Petersen G. M., Gruber S. B., Oddoux C., Ostrer H., Giardiello F. M., Hamilton S. R., Hampel H., Markowitz A., Klimstra D., Jhanwar S., Winawer S., Offit K., Luce M. C., Kinzler K. W., Vogelstein B. Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC. Nat. Genet., 17: 79-83, 1997.[Medline]
  22. Heinimann K., Müllhaupt B., Weber W., Attenhofer M., Scott R. J., Fried M., Martinoli S., Müller H., Dobbie Z. Phenotypic differences in familial adenomatous polyposis based on APC gene mutation status. Gut, 43: 675-679, 1998.[Abstract/Free Full Text]
  23. Giarola M., Stagi L., Presciuttini S., Mondini P., Radice M. T., Sala P., Pierotti M. A., Bertario L., Radice P. Screening for mutations of the APC gene in 66 Italian familial adenomatous polyposis patients: evidence for phenotypic differences in cases with and without identified mutation. Hum. Mutat., 13: 116-123, 1999.[Medline]
  24. Tomlinson I., Rahman N., Frayling I., Mangion J., Barfoot R., Hamoudi R., Seal S., Northover J., Thomas H. J., Neale K., Hodgson S., Talbot I., Houlston R., Stratton M. R. Inherited susceptibility to colorectal adenomas and carcinomas: evidence for a new predisposition gene on 15q14–q22. Gastroenterology, 116: 789-795, 1999.[Medline]
  25. Lu S. L., Kawabata M., Imamura T., Akiyama Y., Nomizu T., Miyazono K., Yuasa Y. HNPCC associated with germline mutation in the TGF-ß type II receptor gene. Nat. Genet., 19: 17-18, 1998.[Medline]
  26. Dobbie Z., D’Orazio D., Attenhofer M., Muller H. Enhanced sensitivity of a modified protein truncation test for exons 1–14 of the adenomatous polyposis coli gene. Biotechniques, 27: 705-708, 1999.[Medline]
  27. Lehnert V., Holzwarth J., Ott M., Thompson A., Demmak S., Foernzler D. A semi-automated system for analysis and storage of SNPs. Hum. Mutat., 17: 243-254,
  28. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16: 1215 1988.[Free Full Text]
  29. Ewing B., Hiller L. D., Wendl M. C., Green P. Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res., 8: 175-185, 1998.[Abstract/Free Full Text]
  30. Ewing B., Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res., 8: 186-194, 1998.[Abstract/Free Full Text]
  31. Gordon D., Abajian C., Green P. Consed: a graphical tool for sequence finishing. Genome Res., 8: 195-202, 1998.[Abstract/Free Full Text]
  32. Nickerson D. A., Tobe V. O., Taylor S. L. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res., 25: 2745-2751, 1997.[Abstract/Free Full Text]
  33. Canzian F., Salovaara R., Hemminki A., Kristo P., Chadwick R. B., Aaltonen L. A., de la Chapelle A. Semiautomated assessment of loss of heterozygosity and replication error in tumors. Cancer Res., 56: 3331-3337, 1996.[Abstract/Free Full Text]
  34. Neugut A. I., Jacobson J. S., Ahsan H., Santos J., Garbowski G. C., Forde K. A., Treat M. R., Waye J. Incidence and recurrence rates of colorectal adenomas: a prospective study. Gastroenterology, 108: 402-408, 1995.[Medline]
  35. Liu H. X., Cartegni L., Zhang M. Q., Krainer A. R. A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat. Genet., 27: 55-58, 2001.[Medline]
  36. Dobbie Z., Spycher M., Hurliman R., Ammann R., Ammann T., Roth J., Muller A., Muller H., Scott R. J. Mutational analysis of the first 14 exons of the adenomatous polyposis coli (APC) gene. Eur. J. Cancer, 30A: 1709-1713, 1994.
  37. Giardiello F. M., Brensinger J. D., Luce M. C., Petersen G. M., Cayouette M. C., Krush A. J., Bacon J. A., Booker S. V., Bufill J. A., Hamilton S. R. Phenotypic expression of disease in families that have mutations in the 5' region of the adenomatous polyposis coli gene. Ann. Intern. Med., 126: 514-519, 1997.[Abstract/Free Full Text]
  38. Yan H., Papadopoulos N., Marra G., Perrera C., Jiricny J., Boland C. R., Lynch H. T., Chadwick R. B., de la Chapelle A., Berg K., Eshleman J. R., Yuan W., Markowitz S., Laken S. J., Lengauer C., Kinzler K. W., Vogelstein B. Conversion of diploidy to haploidy. Nature (Lond.), 403: 723-724, 2000.[Medline]
  39. Dobbie Z., Muller H. Germline mutations in the ß-catenin gene are not associated with the FAP phenotype without an APC mutation. J. Med. Genet., 36: 573-574, 1999.[Free Full Text]
  40. Konishi M., Kikuchi-Yanoshita R., Tanaka K., Muraoka M., Onda A., Okumura Y., Kishi N., Iwama T., Mori T., Koike M., Ushio K., Chiba M., Nomizu S., Konishi F., Utsunomiya J., Miyaki M. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology, 111: 307-317, 1996.[Medline]
  41. Ahuja N., Mohan A. L., Li Q., Stolker J. M., Herman J. G., Hamilton S. R., Baylin S. B., Issa J. P. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res., 57: 3370-3374, 1997.[Abstract/Free Full Text]
  42. Herman J. G., Umar A., Polyak K., Graff J. R., Ahuja N., Issa J. P., Markowitz S., Willson J. K., Hamilton S. R., Kinzler K. W., Kane M. F., Kolodner R. D., Vogelstein B., Kunkel T. A., Baylin S. B. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl. Acad. Sci. USA, 95: 6870-6875, 1998.[Abstract/Free Full Text]
  43. Beck N. E., Tomlinson I. P., Homfray T. F., Frayling I. M., Hodgson S. V., Bodmer W. F. Frequency of germline hereditary non-polyposis colorectal cancer gene mutations in patients with multiple or early onset colorectal adenomas. Gut, 41: 235-238, 1997.[Abstract/Free Full Text]
  44. Joyce J. A., Froggatt N. J., Davies R., Evans D. G., Trembath R., Barton D. E., Maher E. R. Molecular genetic analysis of exons 1 to 6 of the APC gene in non-polyposis familial colorectal cancer. Clin. Genet., 48: 299-303, 1995.[Medline]



This article has been cited by other articles:


Home page
 J. Mol. Diagn.Home page
A. Kaufmann, S. Vogt, S. Uhlhaas, D. Stienen, I. Kurth, H. Hameister, E. Mangold, J. Kotting, E. Kaminsky, P. Propping, et al.
Analysis of Rare APC Variants at the mRNA Level: Six Pathogenic Mutations and Literature Review
J. Mol. Diagn., March 1, 2009; 11(2): 131 - 139.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
E. Castellsague, S. Gonzalez, M. Nadal, O. Campos, E. Guino, M. Urioste, I. Blanco, T. Frebourg, and G. Capella
Detection of APC Gene Deletions Using Quantitative Multiplex PCR of Short Fluorescent Fragments
Clin. Chem., July 1, 2008; 54(7): 1132 - 1140.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
L. S. Rozek, G. Rennert, and S. B. Gruber
APC E1317Q Is Not Associated with Colorectal Cancer in a Population-Based Case-Control Study in Northern Israel.
Cancer Epidemiol. Biomarkers Prev., November 1, 2006; 15(11): 2325 - 2327.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
E. T. Renkonen, P. Nieminen, W. M. Abdel-Rahman, A.-L. Moisio, I. Jarvela, S. Arte, H. J. Jarvinen, and P. Peltomaki
Adenomatous Polyposis Families That Screen APC Mutation-Negative by Conventional Methods Are Genetically Heterogeneous
J. Clin. Oncol., August 20, 2005; 23(24): 5651 - 5659.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
K. M. Murphy, T. Geiger, M. J. Hafez, J. R. Eshleman, C. A. Griffin, and K. D. Berg
A Single Nucleotide Primer Extension Assay to Detect the APC I1307K Gene Variant
J. Mol. Diagn., November 1, 2003; 5(4): 222 - 226.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
O. M. Sieber, H. Lamlum, M. D. Crabtree, A. J. Rowan, E. Barclay, L. Lipton, S. Hodgson, H. J. W. Thomas, K. Neale, R. K. S. Phillips, et al.
Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or ""multiple"" colorectal adenomas
PNAS, February 20, 2002; (2002) 42699199.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
O. M. Sieber, H. Lamlum, M. D. Crabtree, A. J. Rowan, E. Barclay, L. Lipton, S. Hodgson, H. J. W. Thomas, K. Neale, R. K. S. Phillips, et al.
Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or "multiple" colorectal adenomas
PNAS, March 5, 2002; 99(5): 2954 - 2958.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heinimann, K.
Right arrow Articles by Dobbie, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heinimann, K.
Right arrow Articles by Dobbie, Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online