The adenomatous polyposis coli (APC) tumor suppressor

Defects in the APC gene are inarguably linked to the progression of colon cancers that arise both sporadically and through the transmission of germline mutations. Genetic evidence from humans and mouse models suggest that APC is a classic tumor suppressor in that both alleles likely require inactivation for tumor growth to ensue. Nearly all of the mutations, germline and somatic, result in premature termination of the single polypeptide chain, normally consisting of 2843 amino acids. Several definable motifs have now been mapped to the linear amino acid sequence of the APC polypeptide. These include an oligomerization domain, armadillo repeats, binding sites for β-catenin, the human discs large protein, microtubules, and other proteins of unknown function. Inactivation of APC in cancer is likely due to loss of function(s) normally associated with the deleted protein structure.     

Linkage of a variant or attenuated form of adenomatous polyposis coli to the adenomatous polyposis coli (APC) locus.

Adenomatous polyps are an intermediate in the pathway to colon carcinoma. An inherited disorder, familial adenomatous polyposis coli (APC), is characterized by hundreds to thousands of adenomatous polyps. A previously reported family had colon cancer associated with a low average but highly heterogenous number of colonic polyps, this phenotype mapped to the APC locus on 5q. Four new families have been ascertained in which the phenotypic pattern was different from classical polyposis but similar to that of the "prototype" kindred reported earlier. By multilocus linkage analysis, the gene responsible for the disease phenotype was mapped, with a high level of confidence, to the APC locus in two of the four families with the attenuated or variant form of polyposis (AAPC); the results for the two remaining kindreds were inconclusive. A combined maximum LOD score of approximately 7.6 at a recombination fraction of 0 was obtained when the results were summed over the four pedigrees with markers closest to the APC locus. The establishment of genetic linkage in such families may point to the APC locus as having a more significant role in inherited predispositions to colorectal cancer than was previously thought.     

Presymptomatic direct detection of adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis

The recent identification of the familial adenomatous polyposis (FAP) gene (designated as APC) enables conclusive genetic testing of at-risk family members for the specific mutation in families in which the germline gene mutation has been characterized. Presymptomatic molecular diagnosis of FAP was performed by direct direction of mutations in lymphocyte DNA in four families. Each of the families has a different mutation of the APC gene. Twenty-seven offspring of affected individuals (a priori risk of 50%) were tested. Ten of the 27 had already developed clinical features of FAP. Of the remaining seventeen, two had had a negative colon exam at an early age, and nine had never had colon exams (mean age, 12.1±3.1 SD years). Six children from this group (54%) were found to carry their affected parent's mutation. No change in the conventional FAP colon screening regimen is recommended for these children. In contrast, when direct tests indicate that an individual does not have the FAP mutation, we recommended that screening be decreased. Reduction of uncertainty for at-risk FAP family members is an important benefit of genetic testing.     

Regional mapping of 22 microclones around the adenomatous polyposis coli (APC) locus on chromosome 5q

Summary A previously described genomic library constructed from microdissected DNA has been used to generate a large number of probes around the adenomatous polyposis coli (APC) gene at 5q22. A total of 202 clones were hybridised directly onto a somatic cell hybrid panel containing two APC-related interstitial deletions. Of 75 microclones that gave clear hybridisation signals, 22 independent clones mapped into the region common to both deletions. In addition, 4/22 of the markers are conserved in rodent DNA. These clones should provide a valuable resource for screening cDNA libraries and cloning the DNA around the APC gene in yeast artificial chromosomes.This work will be presented in part at HGM11, London.     

Germline mutations of the adenomatous polyposis coli (APC) gene in Algerian familial adenomatous polyposis cohort: first report

Background

Familial adenomatous polyposis (known also as classical or severe FAP) is a rare autosomal dominant colorectal cancer predisposition syndrome, characterized by the presence of hundreds to thousands of adenomatous polyps in the colon and rectum from an early age. In the absence of prophylactic surgery, colorectal cancer (CRC) is the inevitable consequence of FAP. The vast majority of FAP is caused by germline mutations in the adenomatous polyposis coli (APC) tumor suppressor gene (5q21). To date, most of the germline mutations in classical FAP result in truncation of the APC protein and 60% are mainly located within exon 15.

Material and methods

In this first nationwide study, we investigated the clinical and genetic features of 52 unrelated Algerian FAP families. We screened by PCR-direct sequencing the entire exon 15 of APC gene in 50 families and two families have been analyzed by NGS using a cancer panel of 30 hereditary cancer genes.

Results

Among 52 FAP index cases, 36 had 100 or more than 100 polyps, 37 had strong family history of FAP, 5 developed desmoids tumors, 15 had extra colonic manifestations and 21 had colorectal cancer. We detected 13 distinct germline mutations in 17 FAP families. Interestingly, 4 novel APC germline pathogenic variants never described before have been identified in our study.

Conclusions

The accumulating knowledge about the prevalence and nature of APC variants in Algerian population will contribute in the near future to the implementation of genetic testing and counseling for FAP patients.

     

The adenomatous polyposis coli (APC) tumour suppressor--genetics, function and disease

Mutations in the adenomatous polyposis coli (APC) gene are the basis of familial adenomatous polyposis and the majority of sporadic colorectal cancer. APC is expressed in a wide variety of tissues, interacts with the cytoskeleton, is involved in regulating levels of beta-catenin and, most recently, has been shown to bind DNA, suggesting that it may possess a nuclear role. The mutation spectrum implicated in tumorigenesis and its correlation with disease phenotype is well characterized and has contributed to our understanding of important functional domains in APC. Despite these advances, APC continues to provide a fertile subject of research for both colorectal tumorigenesis and cancer in general.     

Germ-line mutations in the first 14 exons of the adenomatous polyposis coli (APC) gene.

The first 14 exons of the APC gene have been screened by the denaturation gradient gel electrophoresis method in 160 unrelated patients with familial adenomatous polyposis coli (APC) syndrome. Four polymorphic variants corresponding to silent mutations not associated with the disease phenotype were observed. Mutations predicted to alter the coding property of the APC gene were observed in 26 patients. All these mutations are expected to lead either to aberrant splicing, to synthesis of a truncated APC protein because of the emergence of a stop codon, or to a change in the translation reading frame. Single-base-pair substitutions were observed on 21 occasions. The most frequent mutation (eight cases) was a C-to-T change which exclusively occurred on the nontranscribed strand within a CG dinucleotide.     

A CA-repeat polymorphism close to the adenomatous polyposis coli (APC) gene offers improved diagnostic testing for familial APC.

Presymptomatic genetic testing for the presence of a mutant allele causing familial adenomatous polyposis coli (APC) has been difficult to perform effectively in the past because DNA markers surrounding the APC gene on chromosome 5q have not been very informative. We report results of genetic linkage studies on both research families and clinical families by using D5S346, a highly polymorphic dinucleotide (CA)-repeat locus 30-70 kb from the APC gene. Linkage analysis with this marker in a large APC pedigree showed an increase of at least 9.0 LOD units, in likelihood of linkage of the disease-causing allele to the APC locus, when compared with the highest LOD score attained with any other closely linked marker. When the first 14 APC families that requested genotypic analysis by the DNA Diagnostic Laboratory at the University of Utah were tested with D5S346, 20 of the 31 at-risk individuals were identified as either carriers or noncarriers of an APC-predisposing allele. We see this marker as an important tool for research studies and for the presymptomatic diagnosis of APC.     

Mechanism of adenomatous polyposis coli (APC)-mediated blockage of long-patch base excision repair

Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase beta (pol-beta) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) (Narayan, S., Jaiswal, A. S., and Balusu, R. (2005) J. Biol. Chem. 280, 6942-6949); however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC-/- cells than in APC+/+ cells. In addition to pol-beta, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-beta and Fen-1, we performed a domain-mapping of APC and found that both pol-beta and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-beta-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Further studies demonstrated that APC blocks 5'-flap endonuclease as well as the 5'-3' exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results, we concluded that APC can have three different effects on the LP-BER pathway. First, APC can block pol-beta-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking the coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking hit-and-run synthesis. These studies will have important implications for APC in DNA damage-induced carcinogenesis and chemoprevention.     

Topoisomerase-I- and Alu-mediated genomic deletions of the APC gene in familial adenomatous polyposis

Germline mutation in the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), a heritable form of colorectal cancer. We have previously reported two novel mutations that delete exons 11 and 14 of the APC gene, respectively, at the cDNA level without any splice junction defects at the genomic level. We describe here the precise breakpoints of the two mutations and the possible mechanisms leading to the genomic rearrangement. The first rearrangement is most likely a topoisomerase-I-mediated non-homologous recombination resulting in a 2-kb deletion that deletes exon 11 of the APC gene. Both 5' and 3' breakpoints have two topoisomerase I recognition sites and runs of pyrimidines within the 10-bp sequences in their vicinity. Further, the 3' breakpoint has an adenine-thymidine-rich region. This is probably the first report of a topoisomerase-I-mediated germline mutation in a tumor suppressor gene. The second rearrangement is most likely an Alu-Alu homologous recombination resulting in a 6-kb deletion encompassing exon 14. The Alu elements at the 5' and 3' breakpoints include the 26-bp core sequence thought to stimulate recombination. In both rearrangements, partial sequences from the long interspersed nuclear element family are in the vicinity of the breakpoints. Other than serving as markers for regions of DNA damage, their precise role in the recombination events, if any, is unclear. Both deletions result in truncated APC proteins missing the beta-catenin- and axin-binding domains, resulting in severe polyposis and cancer.     

Role of adenomatous polyposis coli (APC) and microtubules in directional cell migration and neuronal polarization

In response to extracellular signals during embryonic development, cells undergo directional movements to specific sites and establish proper connections to other cells to form organs and tissues. Cell extension and migration in the direction of extracellular cues is mediated by the actin and microtubule cytoskeletons, and recent results have shed new light on how these pathways are activated by neurotrophins, Wnt or extracellular matrix. These signals lead to modifications of microtubule-associated proteins (MAPs) and point to glycogen synthase kinase (GSK) 3beta as a key regulator of microtubule function during directional migration. This review will summarize these results and then focus on the role of microtubule-binding protein adenomatous polyposis coli (APC) in neuronal polarization and directed migration, and on its regulation by GSK3beta.     

A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth‐related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA‐seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292‐4293‐Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA‐seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene‐based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high‐resolution reference for treating other syndromes associated with supernumerary teeth.     

Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations, small deletions, and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC, using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene, 2065delG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT, SSCP, and conformational sensitive gel electrophoresis (CSGE).     

Linkage disequilibrium predicts physical distance in the adenomatous polyposis coli region.

To test the reliability of linkage-disequilibrium analysis for gene mapping, we compared physical distance and linkage disequilibrium among seven polymorphisms in the adenomatous polyposis coli (APC) region on chromosome 5. Three of them lie within the APC gene, and two lie within the nearby MCC (mutated in colon cancer) gene. One polymorphism lies between the two genes, and one is likely to be 5' of MCC. Five of these polymorphisms are newly reported. All polymorphisms were typed in the CEPH kindreds, yielding 179-205 unrelated two-locus haplotypes. Linkage disequilibrium between each pair of polymorphisms is highly correlated with physical distance in this 550-kb region (correlation coefficient -.80, P < .006). This result is replicated in both the Utah and non-Utah CEPH kindreds. There is a tendency for greater disequilibrium among pairs of polymorphisms located within the same gene than among other pairs of polymorphisms. Trigenic, quadrigenic, three-locus, and four-locus disequilibrium measures were also estimated, but these measures revealed much less disequilibrium than did the two-locus disequilibrium measures. A review of 19 published disequilibrium studies, including this one, shows that linkage disequilibrium nearly always correlates significantly with physical distance in genomic regions > 50-60 kb but that it does not do so in smaller genomic regions. We show that this agrees with theoretical predictions. This finding helps to resolve controversies regarding the use of disequilibrium for inferring gene order. Disequilibrium mapping is unlikely to predict gene order correctly in regions < 50-60 kb in size but can often be applied successfully in regions of 50-500 kb or so in size. It is convenient that this is the range in which other mapping techniques, including chromosome walking and linkage mapping, become difficult.