Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease.

Pear (Pyrus bretschneideri) is a popular fruit worldwide, but the irrational utilization of nitrogen as a fertilizer not only greatly affects the fruit' quality, but also wastes resources and results in serious environmental pollution. To better understand the molecular mechanism in pear responsible for the regulation of nitrate transport and assimilation, RNA-seq was performed on samples collected in response to nitrate treatments. Here, 10,273 differentially expressed genes were obtained and annotated into 49 GO terms, 45 clusters having co-expression trends that involved 18 KEGG-defined significantly overrepresented pathways. The KEGG pathways revealed that 15 unigenes, including one NRT gene, two NR genes, one NiR gene, two GDH genes, six GS genes and three GOGAT genes, were related to nitrogen metabolism and significantly differentially expressed in response to nitrate starvation and a nitrate re-supply treatment. Furthermore, 449 transcription factors belonging to 35 different families were identified during the nitrate treatments. The expression patterns of 14 randomly selected differentially expressed genes were validated by qRT-PCR. This study provides valuable resources for investigating the genetics of the nitrogen metabolic pathways and improving nitrogen utilization efficiency in pear.

Familial adenomatous polyposis is an autosomal dominant hereditary disease characterized by the appearance of hundreds to thousands of colorectal adenomatous polyps; if left untreated, there is nearly a 100% lifetime risk of colorectal cancer. In the present case, adenomatous polyps were observed at 6 years of age. Unlike our previous assumption, adenomatous polyps were detected by colonoscopy at <10 years of age. Considering the clinical importance of early diagnosis, we report this case involving germline adenomatous polyposis coli mutation (c.1958G > C, GenBank: M74088.1) that caused an increase in the isoform without exon 15. Although this isoform has been reported previously, it remains controversial whether the variant is pathogenic or not because it was observed both in patients with familial adenomatous polyposis and in normal controls. Nonetheless, due to quantitative distortion of splice variants in adenomatous polyposis coli transcripts and the early development of adenomatous polyps, we believe that this variant may be pathogenic.

Familial adenomatous polyposis (FAP) is a rare autosomal-inherited disease that highly predisposes to colorectal cancer, characterized by a diffuse duodenal and colorectal polyposis associated with various extradigestive tumors and linked to germline mutations within the APC gene. A French consortium of laboratories involved in APC mutation screening has progressively improved the description of the variation spectrum, inferred functional significance of nontruncating variations, and delineated phenotypic characteristics of the disease. The current version of the UMD-APC database is described here. The total number of variations has risen to 5,453 representing 1,473 distinct variations. The published records initially registered into the database were extended with 3,581 germline variations found through genetic testing performed by the eight licensed laboratories belonging to the French APC network. Sixty six of 149 variations of previously unknown significance have now been classified as (likely) causal or neutral. The database is available on the Internet (http://www.umd.be/APC/) and updated twice per year according to the consensus rules of the network. The UMD-APC database is thus expected to facilitate functional classification of rare synonymous, nonsynonymous, and intronic mutations and consequently improve genetic counseling and medical care in FAP families.

Attenuated familial adenomatous polyposis (AFAP) is a variant of familial adenomatous polyposis with fewer than one hundred colorectal polyps and a later age of onset of the cancer. Here, we report two cases of AFAP within family members. Each patient demonstrated the same novel germ line mutation in exon 15 of the adenomatous polyposis coli (APC) gene and was successfully managed with sulindac after refusal to perform colectomy: a 23-year-old man with incidentally diagnosed gastric adenoma and fundic gland polyps underwent colonoscopy, and fewer than 100 colorectal polyps were found; a 48-year-old woman who happened to be the mother of the 23-year-old man also showed fewer than 100 colorectal polyps on colonoscopy. Genetic analysis revealed a novel frameshift mutation in exon 15 of the APC gene. The deletion of adenine-guanine with the insertion of thymine in c.3833-3834 resulted in the formation of stop codon 1,287 in both patients. The patients were treated with sulindac due to their refusal to undergo colectomy. The annual follow-up upper endoscopy and colonoscopy in the following 2 years revealed significant regression of the colorectal polyps in both patients.

Two main colorectal polyposis syndromes have been described, familial adenomatous polyposis and MUTYH-associated polyposis syndromes. Some polyposis remains unexplained: 20% of adenomatous polyposis and serrated polyposis. The aim of this study was to evaluate in a cohort of patients with unexplained polyposis whether a genetic defect could be detected. Individuals presenting polyposis with more than 40 adenomas or more than 20 serrated polyps (hyperplastic, sessile serrated and mixed), without causative mutation identified, were included. Complementary explorations on APC or MUTYH were performed: search for APC mosaicism, splicing-affecting mutations, large genomic rearrangement of MUTYH. Four genes of Wnt pathway (AXIN2, PPP2R1B, WIF1, SFRP1) and two genes of transforming growth factor-β (TGF-β) pathway (SMAD4, BMPR1A) were screened for germline mutation. Twenty-five patients had an unexplained adenomatous polyposis (familial or sporadic). Five pathogenic mutations were found: four in APC gene (with one case of mosaicism) and one in BMPR1A gene. The exploration of APC mosaicism was better performed from adenoma DNA with high-resolution melting. The screening of the candidate genes did not find any causative mutation. Thirteen individuals had an unexplained serrated polyposis and a frameshift on SMAD4 gene was identified. All mutations were identified in familial cases of polyposis. After new pathological examination, both BMPR1A and SMAD4 cases were found to be associated with a juvenile polyposis while the polyposis was initially described as adenomatous or undetermined. In 17% (6/38) of the patients the causative mutation of the polyposis was identified. Genetic causes were heterogeneous. Sporadic polyposis patients must be considered as potential APC mosaicism. The histological classification of polyposis is strongly important in direct genetic exploration.

In monogenic disorders, the functional evaluation of rare, unclassified variants helps to assess their pathogenic relevance and can improve differential diagnosis and predictive testing. We characterized six rare APC variants in patients with familial adenomatous polyposis at the mRNA level. APC variants c.531 + 5G>C and c.532-8G>A in intron 4, c.1409-2_1409delAGG in intron 10, c.1548G>A in exon 11, and a large duplication of exons 10 and 11 result in a premature stop codon attributable to aberrant transcripts whereas the variant c.1742A>G leads to the in-frame deletion of exon 13 and results in the removal of a functional motif. Mutation c.1548G>A was detected in the index patient but not in his affected father, suggesting mutational mosaicism. A literature review shows that most of the rare APC variants detected by routine diagnostics and further analyzed at the transcript level were evaluated as pathogenic. The majority of rare APC variants, particularly those located close to exon-intron boundaries, could be classified as pathogenic because of aberrant splicing. Our study shows that the characterization of rare variants at the mRNA level is crucial for the evaluation of pathogenicity and underlying mutational mechanisms, and could lead to better treatment modalities.

Familial adenomatous polyposis is an autosomal dominantly inherited syndrome characterized by hundreds or thousands of colorectal polyps and a high risk of colorectal cancer at a young age. Truncating germline mutations in the adenomatous polyposis coli gene are detected in approximately 80 percent of patients with classical familial adenomatous polyposis and in approximately 10 percent of the attenuated familial adenomatous polyposis patients.

We investigated the adenomatous polyposis coli and MUTYH genes mutations in a well-characterized series of 25 unrelated Italian patients with familial adenomatous polyposis.

We characterized the specific adenomatous polyposis coli gene mutation in 10 probands, and identified eight truncating mutations (4 novel and 4 known mutations) and two splicing mutations. One of these, a novel missense mutation in exon 15, activates an exonic splicing enhancer control sequence. Moreover, 11 MUTYH gene mutations have been identified in 7 patients without a dominant family history of polyposis.

This study enlarges the genotype-phenotype correlations of familial adenomatous polyposis and suggests that messenger alterations could be responsible for a subset of familial adenomatous polyposis cases without germ-line adenomatous polyposis coli or MUTYH gene mutations. It also confirms that genotype-phenotype correlations in MUTYH-associated polyposis are very complex.

The dominantly inherited condition familial adenomatous polyposis (FAP) is caused by germline mutations in the APC gene. Finding the causative mutations has great implications for the families. Correlating the genotypes to the phenotypes could help to improve the diagnosis and follow-up of patients.

Mutation screening of APC and the clinical characterization of 96 unrelated FAP patients from the Swedish Polyposis Registry was performed. In addition to generally used mutation screening methods, analyses of splicing-affecting mutations and investigations of the presence of low-frequency mutation alleles, indicating mosaics, have been performed, as well as quantitative real-time polymerase chain reaction to detect lowered expression of APC.

Sixty-one different APC mutations in 81 of the 96 families were identified and 27 of those are novel. We have previously shown that 6 of the 96 patients carried biallelic MUTYH mutations. The 9 mutation-negative cases all display an attenuated or atypical phenotype. Probands with a genotype (codon 1250-1464) predicting a severe phenotype had a median age at diagnosis of 21.8 (range, 11-49) years compared with 34.4 (range, 14-57) years among those with mutations outside this region (P < 0.017). Dense polyposis (> 1000) occurred in 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal cancer among probands was 25% at a mean age of 37.5 years and 29% at a mean age of 46.6 years.

Using a variety of mutation-detection techniques, we have achieved a 100% detection frequency in classical FAP. Probands with APC mutations outside codon 1250-1464, although exhibiting a less-severe phenotype, are at high risk of having a colorectal cancer at diagnosis indicating that age at diagnosis is as important as the severity of the disease for colorectal cancer morbidity.

Specific mutations in the adenomatous polyposis coli (APC) gene can lead to an attenuated form of familial adenomatous polyposis (AFAP). Although AFAP mutation carriers have a 69% risk of colorectal cancer by age 80, clinical recognition remains a challenge in some cases because they present with few colonic adenomas and are difficult to distinguish clinically from patients with sporadic polyps.

Family relationships were established using family history reports, the Utah Population Database, and the public records of the Mormon Church. Genetic analysis of representative family members was performed using a 10,000 single nucleotide polymorphism array platform. Colonoscopy data were available on 120 individuals with the AFAP mutation.

Two large AFAP kindreds with the identical APC disease-causing mutation (c.426_427delAT) were linked to a founding couple who came to America from England around 1630. Genetic analysis showed that the 2 families share a conserved haplotype of 7.17 Mbp surrounding the mutant APC allele. The data show that 36.6% of the mutation-positive family members have fewer than 10 colonic adenomatous polyps, and 3 (6.8%) of these individuals were diagnosed with colorectal cancer.

In view of the apparent age of this mutation, a notable fraction of both multiple-adenoma patients and perhaps even colon cancer cases in the United States could be related to this founder mutation. The colon cancer risk associated with the mutation makes genetic testing of considerable importance in patients with a personal or family history of either colonic polyps or cancer at a young age.

A small fraction of families with familial adenomatous polyposis (FAP) display an attenuated form of FAP (AFAP). We aimed to assess the presence of germline mutations in the MUTYH and adenomatous polyposis coli (APC) genes in AFAP families and to compare the clinical features between the two causative genes. Families with clinical AFAP were selected from the Dutch Polyposis Registry according to the following criteria: (a) at least two patients with 10-99 adenomas diagnosed at age >30 years or (b) one patient with 10-99 adenomas at age >30 years and a first-degree relative with colorectal cancer (CRC) with a few adenomas, and, applying for both criteria, no family members with more than 100 polyps before the age of 30 years. All probands were screened for germline mutations in the APC and MUTYH genes. Twenty-five of 315 Dutch families with FAP (8%) met our criteria for AFAP. These families included 146 patients with adenomas and/or CRC. Germline APC mutations were identified in nine families and biallelic MUTYH mutations in another nine families. CRC was identified at a mean age of 54 years (range 24-83 years) in families with APC and at 50 years (range 39-70 years) in families with MUTYH (p = 0.29). APC and biallelic MUTYH mutations are responsible for the majority of AFAP families. Based on our results and those reported in the literature, we recommend colonoscopy once every 2 years in AFAP families, starting surveillance from the late teens in APC mutation carriers and from age 20-25 years in biallelic MUTYH mutation carriers.

One third of families with classical adenomatous polyposis (FAP), and a majority of those with attenuated FAP (AFAP), remain APC mutation-negative by conventional methods. Our purpose was to clarify the genetic basis of polyposis and genotype-phenotype correlations in such families.

We studied a cohort of 29 adenomatous polyposis families that had screened APC mutation-negative by the protein truncation test, heteroduplex analysis, and exon-specific sequencing. The APC gene was investigated for large genomic rearrangements by multiplex ligation-dependent probe amplification (MLPA), and for allelic mRNA expression by single nucleotide primer extension (SNuPE). The AXIN2 gene was screened for mutations by sequencing.

Four families (14%) showed a constitutional deletion of the entire APC gene (three families) or a single exon (one family). Seven families (24%) revealed reduced or extinct mRNA expression from one APC allele in blood, accompanied by loss of heterozygosity in the APC region in six (75%) of eight tumors. In 15 families (52%), possible APC involvement could be neither confirmed nor excluded. Finally, as detailed elsewhere, three families (10%) had germline mutations in genes other than APC, AXIN2 in one family, and MYH in two families.

"APC mutation-negative" FAP is genetically heterogeneous, and a combination of MLPA and SNuPE is able to link a considerable proportion (38%) to APC. Significant differences were observed in clinical manifestations between subgroups, emphasizing the importance of accurate genetic and clinical characterization for the proper management of such families.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, the relevance of rare exonic single-base substitutions at nucleotide positions close to splice sites that are predicted to result in missense or silent (SNP) variants or substitutions in introns at splice-site positions that are not highly conserved has not been systematically examined in FAP patients. In 34 index patients, we identified 26 different heterozygous single-base substitutions at or close to the splice sites. We characterized five exonic mutations in exon 4 (c.423G>T), exon 14 (c.1956C>T, c.1957A>G, and c.1957A>C), and exon 15 (c.1959G>A) by transcript analysis and by splice-prediction programs (BDGP, SpliceSiteFinder, and ESEfinder). The splicing patterns of these variants were compared to those of 16 different substitutions at highly or less-conserved intronic splice-site positions, and to normal controls. In addition, we analyzed cosegregation of the variants with affected family members and examined the genotype-phenotype correlation. We could demonstrate that the four unclear variants in exon 4 and 14 that are predicted to result in missense or silent mutations in fact lead to complete exon skipping due to aberrant splicing; one possible explanation for this observed effect might be the disruption of exonic splicing enhancer (ESE) motifs. In contrast, the substitution at the first position of exon 15 seems to actually be a silent variant. We present the first systematic evaluation of different single-base substitutions in APC at or close to splice sites at transcript level. We show that the consequence of exonic mutations cannot be evaluated only by the predicted change in amino acid sequence but rather by the change at DNA level. The functional analysis of variants with unknown pathogenic effect plays an important role in increasing the mutation detection rate and achieving validation of predictive testing.

Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions/deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene.

In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation - dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction).

A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients.

Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations.

The adenomatous polyposis coli (APC) protein is a tumor suppressor frequently involved in the development of inherited and sporadic colon cancers. Somatic mutations of the APC gene are found in 80% of all colon cancers. Inherited mutations result in familial adenomatous polyposis (FAP) as well as an attenuated form of this syndrome. FAP is characterized by the early age onset of hundreds to thousands of colonic adenomatous polyps and a virtual certainty of colon cancer unless the colon is removed. The attenuated form of FAP (AFAP) is characterized by fewer adenomas, later onset of adenomas and cancer, and a decreased lifetime cancer risk. We report a 37-year-old man with a history of more than 50 colonic adenomatous polyps, located predominately in the right colon. An insertion of a single thymidine between the second and third base pairs of intron 4 of the APC gene was identified (c.531+2_531+3insT). Monoallelic hybrid cells harboring a single copy of human chromosome 5 were generated from patient lymphoblasts. Sequencing of the APC cDNA product from these cells revealed a single RNA transcript with aberrant splicing in the mutant mRNA whereby exon 4 is deleted. The translational reading frame is shifted after codon 140 and a translational stop is generated predicting a truncated protein of 147 amino acids, thus indicating that the intronic mutation is disease causing. The lack of a secondary transcript from the mutant allele suggests that incomplete exon skipping is not the molecular mechanism behind the attenuated phenotype.

Familial adenomatous polyposis (FAP) is an autosomal dominant condition characterized by the development of hundreds to thousands of colorectal adenomatous polyps. In addition to the classic form, there is also attenuated polyposis (attenuated adenomatous polyposis coli; AAPC), which is characterized by a milder phenotype. FAP/AAPC is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Very recently, germline mutations in the base-excision repair gene MYH have been associated with recessive inheritance of multiple colorectal adenomas in a subset of patients. APC pathogenic alterations are mostly (>95%) represented by frameshift or nonsense mutations leading to the synthesis of a truncated protein. We identified 20 APC truncating mutation carriers out of 30 FAP/AAPC patients from different Italian kindreds. In the remaining 10 patients, we searched for alterations other than truncating mutations by enzymatic mutation detection, real-time quantitative RT-PCR, and genotyping of polymorphic markers encompassing the APC locus. Moreover, to assess whether mutations of genes interacting with APC can substitute or act in association with APC alterations, we sequenced both CTNNB1 (beta-catenin) and CDH1 (E-cadherin) genes. No CTNNB1 or CDH1 mutations were found. On the contrary, four patients showed a reduced APC gene expression compared with healthy subjects. In three of the four cases, genotyping results were compatible with a constitutive allelic deletion. In one case this conclusion was confirmed by haplotype segregation analysis. Our results support the notion that FAP/AAPC can result from APC constitutive haploinsufficiency, with gene deletion being a possible cause of reduced gene expression.

The attenuated form of familial adenomatous polyposis coli (AAPC) is associated with mutations in the adenomatous polyposis coli (APC) gene which cluster in the 5' region of the gene. It has been proposed that a 'genotype-phenotype boundary' exists at codons 159-163, and mutations that are 5' of this boundary will produce AAPC. Herein we document a three-generation family with an exon 3 mutation well to the 5' side of the proposed boundary, in which two affected individuals have had, in their 40s, a profuse form of familial adenomatous polyposis coli. We conclude that the codon 159-163 'boundary' is indicative rather than definitive. These two patients also had postoperative intra-abdominal adhesions, severely so in one.

Some truncating mutations of the APC tumor suppressor gene are associated with an attenuated phenotype of familial adenomatous polyposis coli (AAPC). This work demonstrates that APC alleles with 5' mutations produce APC protein that down-regulates beta-catenin, inhibits beta-catenin/T cell factor-mediated transactivation, and induces cell-cycle arrest. Transfection studies demonstrate that cap-independent translation is initiated internally at an AUG at codon 184 of APC. Furthermore, APC coding sequence between AAPC mutations and AUG 184 permits internal ribosome entry in a bicistronic vector. These data suggest that AAPC alleles in vivo may produce functional APC by internal initiation and establish a functional correlation between 5' APC mutations and their associated clinical phenotype.

Adenine paired with 8-hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products.

An aberrant size of the beta type nuclear form transcript was detected in a lung cancer cell line, VMRC-LCD, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5'-side of the native ORF in the beta type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5'-untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT-PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (approximately 30) than that of the IVS1+5G transcript.

The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells.

Androgen receptor (AR) gene mutations have been shown to cause androgen insensitivity syndrome with altered sexual differentiation in XY individuals, ranging from a partial insensitivity with male phenotype and azoospermia to a complete insensitivity with female phenotype and the absence of pubic and axillary sexual hair after puberty. In this study we present an 11-yr-old XY girl, with clinical manifestations peculiar for impaired androgen biological action, including female phenotype, blind-ending vagina, small degree of posterior labial fusion, and absence of uterus, fallopian tubes, and ovaries. At the time of the diagnosis the patient had a FSH/LH ratio according to the puberal stage, undetectable 17beta-estradiol, and high levels of testosterone (80.1 ng/mL). After bilateral gonadectomy, performed at the age of 11 yr, histological examination showed small embryonic seminiferous tubules containing prevalently Sertoli cells and occasional spermatogonia together with abundant fibrous tissue. Molecular study of the patient showed a guanine to thymine transversion in position +5 of the donor splice site in the junction between exon 6 and intron 6 of the AR gene. The result of RT-PCR amplification of the AR messenger ribonucleic acid from cultured genital skin fibroblasts of the patient suggests that splicing is defective, and intron 6 is retained in most of the receptor messenger ribonucleic acid molecules. We show by immunoblotting that most of the expressed protein lacks part of the C-terminal hormone-binding domain, and a small amount of normal receptor is observed. This is probably responsible for the reduced binding capacity in genital skin fibroblasts of the patient. The molecular basis of the alteration in this case is a novel, uncommon mutation, leading to a phenotype indicative of a partial androgen insensitivity syndrome, Quigley's grade 5.

Adenomatous polyposis coli (APC) gene transcripts skipping exon 14 in combination with the alternatively spliced exons 9 and 10A contribute to the heterogeneity of physiological APC mRNA isoforms. Here we report on a novel genotype-phenotype correlation in familial adenomatous polyposis (FAP) with early onset of disease and malignancy due to an APC exon 14 splice defect. Compared to controls, two affected individuals of a FAP kindred presented with a significantly distorted APC mRNA isoform pattern in B lymphocytes. As a result of an A-->G transition in the canonical AG-splice acceptor dinucleotide of exon 14, expression levels of all APC mRNA isoforms without exon 14 were dramatically increased and those with exon 14 were simultaneously decreased. Skipping of exon 14 is a physiological event also seen in nonmalignant cells, which results in a frameshift to produce low-molecular-weight APC proteins. Western blot analysis of the patients' lymphoblastoid B cells revealed the identification of intracellularly stable APC protein isoforms with an Mr of 55-67 kDa and, thus, the first demonstration of APC proteins encoded by exon 14-skipped transcripts. We postulate that the quantitatively imbalanced expression of these physiological APC light chains represents a novel pathogenetic mechanism associated with predisposition to FAP.

A family is presented with attenuated familial adenomatous polyposis of variable phenotype. The clinical features range from sparse right-sided polyposis and cancer in the proximal colon at the age of 34 to pan-colonic polyposis and cancer at the age of 68. Rectal sparing is common to all affected members. Heteroduplex analysis detected bands of altered mobility in exon 9 of the APC gene in all affected family members. Subsequently, a frameshift mutation was found in the alternatively spliced region of exon 9 at codon 398 which resulted in a stop signal 4 codons downstream. Alternatively spliced transcripts that delete the mutation were readily amplified from normal colonic mucosa and therefore create a mechanism for the attenuated phenotype seen in this family.

Most inherited mutant alleles of the adenomatosis polyposis coli gene (APC) cause the appearance of large numbers of colon polyps, the familial polyposis syndrome. (These mutant alleles are designated APCp alleles.) A subset of APC mutations, the attenuated or APC(AP) alleles, predispose to only a few colon polyps. This leads to the hypothesis that if mutation of the inherited normal allele is rate limiting in polyp development, the increased number of polyps associated with the APCp allele indicates that the frequency of mutations that can lead to polyp formation is higher among APCp carriers than among APC(AP) carriers. We have previously suggested that the APC protein might modulate the frequency of mutations, such as loss of heterozygosity (LOH), necessary for colon polyp formation. We thus reasoned that tumours from patients who carry an APC(AP) allele might show a reduced frequency of LOH compared with tumours from patients who carry an APCp allele. Loss of AAPC mutant alleles is designated as LOH(AP). Screening of tumours from APC(AP) carriers revealed a reduction of LOH compared with that of an unselected group of polyposis patients. In fact, no loss of the inherited APC(N) allele was observed, although sequencing showed that the inherited APC(N) allele had frequently undergone point mutations and small deletions in the tumours. A low frequency loss of the inherited APC(AP) allele was seen. These findings support the suggestion that the APC(AP) allele has residual gene activity and that this activity modulates the spectrum and frequency of mutations that lead to adenoma formation.

Chain-terminating germline APC mutations are responsible for adenomatous polyposis coli (APC). In the present work, we tested the hypothesis that germline APC mutations may be present in some patients with a milder phenotype, i.e., multiple synchronous colorectal adenomas. Eighteen patients with 3 or more colorectal adenomas at endoscopy (within a 6-month period) were ascertained from a series of subjects undergoing endoscopic examination. Their blood DNAs were analysed for the presence of germline mutations in the APC coding region by single-strand polymorphism analysis. Ten unrelated polyp-free subjects and 101 unrelated APC patients were used as controls in the molecular analyses. Five of the eighteen patients carried novel germline APC variants or rare polymorphisms. These were various in site (from the splice acceptor site of intron 7 to the end of exon 15) and type (splice-site, missense, and chain-terminating mutations). Only one of ten polyp-free individuals carried a silent APC variant and none of these variants was found in the 101 APC controls. A first- or second-degree family history of colorectal cancer was reported by 4 of the 5 patients carrying a germline APC variant. In conclusion, novel APC germline variants were detected in patients with multiple synchronous adenomas. This suggests that the development of sporadic adenomas, in some instances, is associated with the presence of minor germline variants of the APC gene and that the spectrum of germline APC functional mutations may be larger than previously thought.

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.

Analysis of genotype-phenotype correlations in familial adenomatous polyposis (FAP) patients demonstrated that the phenotypic heterogeneity of FAP is partly related to the mutation site. We investigated the molecular basis for the difference in severity of colorectal disease observed comparing FAP patients from two kindreds with neighbouring germline mutations in exon 9 of the APC gene. Patients from one kindred presented with a attenuated form of FAP, characterized by a low number of colorectal adenomas (up to 22). In FAP patients from this kindred, the APC gene mutation was localized at codon 367, in the portion of exon 9 that is alternately spliced. This is expected to result in the splicing-out of the mutation site in a fraction of mRNA molecules and in the residual production of wild-type transcripts from the mutant APC allele. Patients from the other kindred manifested a FAP phenotype characterized by hundreds of colorectal adenomas (320 to > 500). In these patients, the APC gene mutation abolished the donor site of exon 9a, used in both alternately spliced isoforms of the exon. The analysis of the relative levels of mutant and wild-type transcripts in unaffected colonic mucosa demonstrated that the mutant allele was not expressed. The model offered by our FAP patients with neighbouring exon 9 APC mutations supports the view that in addition to the mutation site, the type of mutation and transcript dosage effects contribute to the heterogeneity of disease phenotypes.

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 beta-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.

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. We screened the entire coding region of the APC gene for mutations in an unselected series of 105 Dutch FAP kindreds. For the analysis of exons 1-14, we employed the GC-clamped denaturing gradient gel electrophoresis (DGGE), while the large exon 15 was examined using the protein truncation test. Using this approach, we identified 65 pathogenic mutations in the above 105 apparently unrelated FAP families. The mutations were predominantly either frameshifts (39/65) or single base substitutions (18/65), resulting in premature stop codons. Mutations that would predict abnormal RNA splicing were identified in seven cases. In one of the families, a nonconservative amino acid change was found to segregate with the disease. In spite of the large number of APC mutations reported to date, we identified 27 novel germline mutations in our patients, which reiterates the great heterogeneity of the mutation spectrum in FAP. In addition to the point mutations identified in our patients, structural rearrangements of APC were found in two pedigrees, by Southern blot analysis. The present study indicates that the combined use of DGGE, protein truncation test, and Southern blot analysis offers an efficient strategy for the presymptomatic diagnosis of FAP by direct mutation detection. We found that the combined use of the currently available molecular approaches still fails to identify the underlying genetic defect in a significant subset of the FAP families. The possible causes for this limitation are discussed.

The identification of germline mutations in a large number of clinically well-characterised patients with familial adenomatous polyposis (FAP) has allowed the unravelling of several genotype-phenotype relationships that can now be interpreted in the light of the structure and functional domains of the adenomatous polyposis coli (APC) protein. An attenuated phenotype has been found to be associated with mutations at the 5' end of the gene, while a severe clinical expression was found in patients with the most common mutation at codon 1309. So far, only few mutations in the 3' half of the gene have been published. We report on two families with a rather mild phenotype due to a frameshift mutation at codon 1597. These families may represent a clue for defining a 5' border for the occurrence of a second region of attenuated FAP that is localised in the 3' part of the APC gene. We propose a model to explain the relationship between the severity of the disease and the size of the mutant APC protein.

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.

Nine new causative mutations and seven previously characterised mutations of the APC gene of patients with familial adenomatous polyposis (FAP) were analysed for any genotype-phenotype correlations. The only clear genotype-phenotype correlation found was between the position of the mutation site and the presence or absence of congenital hypertrophy of the retinal pigment epithelium (CHRPE). A more distal mutation site was associated with an earlier age of onset of symptoms and a larger number of colonic polyps, but a notable amount of intrafamilial variation was observed.

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer caused by germline mutations in the adenomatous polyposis coli (APC) gene located on chromosome segment 5q21-q22. We detected a germline rearrangement of the APC gene in a Dutch FAP family by screening genomic DNA samples with APC cDNA probes. Subsequent molecular and cytogenetic studies revealed a constitutional reciprocal translocation t(5;10)(q22;q25) that resulted in the disruption of the APC gene. Southern blot and polymorphic marker analysis indicated that part of the APC gene had been deleted. Analysis of the APC protein product indicated that the translocation breakpoint did not lead to the formation of a detectable truncated APC protein but apparently resulted in a null allele. Evaluation of the clinical phenotypes in the patients suggested that they exhibited features of an unusual form of FAP characterized by a slightly delayed age of onset of colorectal cancer and a reduced number of colorectal polyps. The latter were mainly sessile and were located predominantly in the proximal colon. To our knowledge, this is the first description of FAP caused by a reciprocal translocation disrupting the APC gene.

An enormous number of germline and somatic mutations have been identified in the APC tumor suppressor gene. Nearly all of these mutations result in premature polypeptide chain termination, but the consequences to APC protein function are unknown. Recent advances, including the identification of an oligomerization domain, the localization of several beta-catenin binding sites, some of which down-regulate beta-catenin in vivo, and the identification of a microtubule-binding domain in the carboxy-terminal region of APC, are beginning to provide some clues.

Heterogeneity among and within FAP pedigrees for the age of symptom onset and the age at death from colorectal cancer was studied in a sample of 583 patients of the Italian Polyposis Registry. The among pedigree variation was largely explained by clustering of families in two groups, 'early FAP' (most colorectal cancer deaths below 45 years of age) and 'late FAP' families (most deaths above age 45). The within-family variation was explained by a marked phenomenon of anticipation (15 years per generation, on the average), possibly not due to ascertainment bias. We then considered the pedigrees with identified mutation in the APC gene. Six families shared a common deletion at codon 1309 and showed the early FAP phenotype. Two families shared a mutation at codon 1061 and revealed the late FAP phenotype. Another two families (codons 453 and 302) clustered with the late FAP group, whereas a family with mutation at codon 835 clustered with the early FAP group. We suggest that there are at least two classes of mutations in the APC gene with different consequences at the phenotypic level. It seems that there are several critical points within the APC protein sequence at which truncation causes a more aggressive disease than truncation at other points.