Germline mutations in the 3′ part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli

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. Received: 18 June 1996 / Revised: 8 July 1996     

The identical 5' splice-site acceptor mutation in five attenuated APC families from Newfoundland demonstrates a founder effect

Inherited mutations of the APC gene predispose carriers to multiple adenomatous polyps of the colon and rectum and to colorectal cancer. Mutations located at the extreme 5' end of the APC gene, however, are associated with a less severe disease known as attenuated adenomatous polyposis coli (AAPC). Many individuals with AAPC develop relatively few colorectal polyps but are still at high risk for colorectal cancer. We report here the identification of a 5' APC germline mutation in five separately ascertained AAPC families from Newfoundland, Canada. This disease-causing mutation is a single basepair change (G to A) in the splice-acceptor region of APC intron 3 that creates a mutant RNA without exon 4 of APC. The observation of the same APC mutation in five families from the same geographic area demonstrates a founder effect. Furthermore, the identification of this germline mutation strengthens the correlation between the 5' location of an APC disease-causing mutation and the attenuated polyposis phenotype.     

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.     

Genotype-phenotype correlations in attenuated adenomatous polyposis coli.

Germ-line mutations of the tumor suppressor APC are implicated in attenuated adenomatous polyposis coli (AAPC), a variant of familial adenomatous polyposis (FAP). AAPC is recognized by the occurrence of <100 colonic adenomas and a later onset of colorectal cancer (age >40 years). The aim of this study was to assess genotype-phenotype correlations in AAPC families. By protein-truncation test (PTT) assay, the entire coding region of the APC gene was screened in affected individuals from 11 AAPC kindreds, and their phenotypic differences were examined. Five novel germ-line APC mutations were identified in seven kindreds. Mutations were located in three different regions of the APC gene: (1) at the 5' end spanning exons 4 and 5, (2) within exon 9, and (3) at the 3' distal end of the gene. Variability in the number of colorectal adenomas was most apparent in individuals with mutations in region 1, and upper-gastrointestinal manifestations were more severe in them. In individuals with mutations in either region 2 or region 3, the average number of adenomas tended to be lower than those in individuals with mutations in region 1, although age at diagnosis was similar. In all AAPC kindreds, a predominance of right-sided colorectal adenomas and rectal polyp sparing was observed. No desmoid tumors were found in these kindreds. Our data suggest that, in AAPC families, the location of the APC mutation may partially predict specific phenotypic expression. This should help in the design of tailored clinical-management protocols in this subset of FAP patients.     

Hereditary desmoid disease due to a frameshift mutation at codon 1924 of the APC gene.

Desmoid tumors are slowly growing fibrous tumors highly resistant to therapy and often fatal. Here, we report hereditary desmoid disease (HDD), a novel autosomal dominant trait with 100% penetrance affecting a three-generation kindred. Desmoid tumors are usually a complication of familial adenomatous polyposis, a predisposition to the early development of premalignant adenomatous polyps in the colorectum due to chain-terminating mutations of the APC gene. In general, one or more members in approximately 10% of the FAP families manifest desmoid tumors. Affected individuals from the HDD kindred are characterized by multifocal fibromatosis of the paraspinal muscles, breast, occiput, arms, lower ribs, abdominal wall, and mesentery. Osteomas, epidermal cysts, and other congenital features were also observed. We show that HDD segregates with an unusual germ-line chain-terminating mutation at the 3' end of the APC gene (codon 1924) with somatic loss of the wild-type allele leading to tumor development.     

Role of heterozygous APC mutation in niche succession and initiation of colorectal cancer--a computational study

Mutations in the adenomatous polyposis coli (APC) gene are found in most colorectal cancers. They cause constitutive activation of proliferative pathways when both alleles of the gene are mutated. However studies on individuals with familial adenomatous polyposis (FAP) have shown that a single mutated APC allele can also create changes in the precancerous colon crypt, like increased number of stem cells, increased crypt fission, greater variability of DNA methylation patterns, and higher somatic mutation rates. In this paper, using a computational model of colon crypt dynamics, we evolve and investigate a hypothesis on the effect of heterozygous APC mutation that explains these different observations. Based on previous reports and the results from the computational model we propose the hypothesis that heterozygous APC mutation has the effect of increasing the chances for a stem cell to divide symmetrically, producing two stem cell daughters. We incorporate this hypothesis into the model and perform simulation experiments to investigate the consequences of the hypothesis. Simulations show that this hypothesis links together the changes in FAP crypts observed in previous studies. The simulations also show that an APC(+/-) stem cell gets selective advantages for dominating the crypt and progressing to cancer. This explains why most colon cancers are initiated by APC mutation. The results could have implications for preventing or retarding the onset of colon cancer in people with inherited or acquired mutation of one APC allele. Experimental validation of the hypothesis as well as investigation into the molecular mechanisms of this effect may therefore be worth undertaking.     

Exclusion of the APC gene as the cause of a variant form of familial adenomatous polyposis (FAP)

Familial adenomatous polyposis (FAP) is a premalignant disease inherited as an autosomal dominant trait, characterized by hundreds to thousands of polyps in the colorectal tract. Recently, the syndrome has been shown to be caused by mutations in the APC (adenomatous polyposis coli) gene located on chromosome 5q21. We studied two families that both presented a phenotype different than that of the classical form of FAP. The most important findings observed in these two kindreds are (a) low and variable number of colonic polyps (from 5 to 100) and (b) a slower evolution of the disease, with colon cancer occurring at a more advanced age than in FAP in spite of the early onset of intestinal manifestations. To determine whether mutations of the APC gene are also responsible for this variant syndrome, linkage studies were performed by using a series of markers both intragenic and tightly linked to the APC gene. The results provide evidence for exclusion of the APC gene as the cause of the variant form of polyposis present in the two families described.     

Inherited colorectal polyposis and cancer risk of the APC I1307K polymorphism.

Germ-line and somatic truncating mutations of the APC gene are thought to initiate colorectal tumor formation in familial adenomatous polyposis syndrome and sporadic colorectal carcinogenesis, respectively. Recently, an isoleucine-->lysine polymorphism at codon 1307 (I1307K) of the APC gene has been identified in 6%-7% of the Ashkenazi Jewish population. To assess the risk of this common APC allelic variant in colorectal carcinogenesis, we have analyzed a large cohort of unselected Ashkenazi Jewish subjects with adenomatous polyps and.or colorectal cancer, for the APC I1307K polymorphism. The APC I1307K allele was identified in 48 (10.1%) of 476 patients. Compared with the frequency in two separate population control groups, the APC I1307K allele is associated with an estimated relative risk of 1.5-1.7 for colorectal neoplasia (both P=.01). Furthermore, compared with noncarriers, APC I1307K carriers had increased numbers of adenomas and colorectal cancers per patient (P=.03), as well as a younger age at diagnosis. We conclude that the APC I1307K variant leads to increased adenoma formation and directly contributes to 3%-4% of all Ashkenazi Jewish colorectal cancer. The estimated relative risk for carriers may justify specific clinical screening for the 360,000 Americans expected to harbor this allele, and genetic testing in the setting of long-term-outcome studies may impact significantly on colorectal cancer prevention in this population.     

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.     

A Highlights from MBoC Selection: APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.     

APC binds intermediate filaments and is required for their reorganization during cell migration

Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.     

APC mutation in the alternatively spliced region of exon 9 associated with late onset familial adenomatous polyposis

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.     

The intestinal epithelium and its neoplasms: genetic,cellular and tissue interactions.

The Min (multiple intestinal neoplasia) strain of the laboratory mouse and its derivatives permit the fundamental study of factors that regulate the transition between normal and neoplastic growth. A gene of central importance in mediating these alternative patterns of growth is Apc, the mouse homologue of the human adenomatous polyposis coli (APC) gene. When adenomas form in the Min mouse, both copies of the Apc gene must be inactivated. One copy is mutated by the nonsense Apc allele carried in heterozygous form in this strain. The other copy can be silenced by any of several mechanisms. These range from loss of the homologue bearing the wild-type Apc allele; to interstitial deletions surrounding the wild-type allele; to intragenic mutation, including nonsense alleles; and finally, to a reduction in expression of the locus, perhaps owing to mutation in a regulatory locus. Each of these proposed mechanisms may constitute a two-hit genetic process as initially posited by Knudson; however, apparently the two hits could involve either a single locus or two loci. The kinetic order for the transition to adenoma may be still higher than two, if polyclonal adenomas require stronger interactions than passive fusion. The severity of the intestinal neoplastic phenotype of the Min mouse is strongly dependent upon loci other than Apc. One of these, Mom1, has now been rigorously identified at the molecular level as encoding an active resistance conferred by a secretory phospholipase. Mom1 acts locally within a crypt lineage, not systemically. Within the crypt lineage, however, its action seems to be non-autonomous: when tumours arise in Mom1 heterozygotes, the active resistance allele is maintained in the tumour (MOH or maintenance of heterozygosity). Indeed, the secretory phospholipase is synthesized by post-mitotic Paneth cells, not by the proliferative cells that presumably generate the tumour. An analysis of autonomy of modifier gene action in chimeric mice deserves detailed attention both to the number of genetic factors for which an animal is chimeric and to the clonal structure of the tissue in question. Beyond Mom1, other loci can strongly modify the severity of the Min phenotype. An emergent challenge is to find ways to identify the full set of genes that interact with the intestinal cancer predisposition of the Min mouse strain. With such a set, one can then work, using contemporary mouse genetics, to identify the molecular, cellular and organismal strategies that integrate their functions. Finally, with appropriately phenotyped human families, one can investigate by a candidate approach which modifying factors influence the epidemiology of human colon cancer. Even if a candidate modifier does not explain any of the genetic epidemiology of colon cancer in human populations, modifier activities discovered by mouse genetics provide candidates for chemopreventive and/or therapeutic modalities in the human.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.