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.     

New insights into allelic diversity of a phosphoenolpyruvate carboxylase in the conifer Picea abies (L.) Karst.

Two genomic full-length alleles of a phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were isolated and analysed in the gymnosperm Norway spruce [Picea abies (L.) Karst.]. Mendelian segregation analysis confirmed that the two alleles belong to the same DNA gene locus. Southern analysis was performed using exon and intron probes, separately. A small gene family was discovered when exon probes were used. The isolated gene locus was termed PEPC-1 and found to exhibit the largest PEPC genomic sequence characterised so far in the plant kingdom. This is due to comparatively long intron sequences, and to an additional intron in the 3'-untranslated region. Sequence homology of the two full-length PEPC-1 alleles is 99.8%. From exon variation, one single exchange of an amino acid was deduced. The introns harboured polymorphic regions that were analysed for variability among three different spruce individuals. Within-PEPC-1 genotyping revealed that all three individuals could be unambiguously distinguished from each other. The results are discussed with respect not only to exon/intron site evolution among alleles but also to particular genomic features of a gymnosperm PEPC in comparison to PEPC in angiosperms.     

Genomic rearrangements of the APC tumor-suppressor gene in familial adenomatous polyposis

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in the hereditary colorectal cancer syndrome familial adenomatous polyposis (FAP). Almost all APC mutations that have been identified are single-nucleotide alterations, small insertions, or small deletions that would truncate the protein product of the gene. No well-characterized intragenic rearrangement of APC has been described, and the prevalence of this type of mutation in FAP patients is not clear. We screened 49 potential FAP families and identified 26 different germline APC mutations in 30 families. Four of these mutations were genomic rearrangements resulting from homologous and nonhomologous recombinations mediated by Alu elements. Two of these four rearrangements were complex, involving deletion and insertion of nucleotides. Of these four rearrangements, one resulted in the deletion of exons 11 and 12 and two others resulted in either complete or partial deletion of exon 14. The fourth rearrangement grossly altered the sequence within intron 14. Although this rearrangement did not affect any coding sequence of APC at the genomic DNA level, it caused inappropriate splicing of exon 14. These rearrangements were initially revealed by analyzing cDNAs and could not have been identified by using mutation detection methods that screened each exon individually. The identification of a rearrangement that did not alter any coding exons yet affected the splicing further underscores the importance of using cDNA for mutation analysis. The identification of four genomic rearrangements among 30 mutations suggests that genomic rearrangements are frequent germline APC mutations.     

Rapid changes in the exon/intron structure of a mammalian thrombin inhibitor gene

The genomic organization of the heparin cofactor II (HCII) gene from rat and mouse was investigated and compared with their human counterpart. The genes share a common core structure consisting of five exons interrupted by four introns, but the mouse and rat gene reveal individual additional features. A unique differentially spliced exon is present in the 5'-untranslated region of the rat gene, which most probably has arisen de novo by point mutations in intronic sequences of the ancestor gene. In the mouse HCII gene, a novel intron/exon boundary has been created due to the presence of an additional DNA segment, which simultaneously provides a 3'-splice site and a polypyrimidine stretch leading to an alternatively used exon of increased size. Our data suggest that, in contrast to most other mammalian genes, the exon/intron pattern of the gene coding for HCII is in dynamic evolution.     

APC germ-line mutations in southern Spanish patients with familial adenomatous polyposis: genotype-phenotype correlations and identification of eight novel mutations

Familial adenomatous polyposis (FAP) is a disease characterized by the presence of hundreds of adenomatous polyps in the colon and rectum which, if not treated, develop into colorectal cancer. FAP is an autosomal dominantly inherited disorder caused by mutation in the APC gene. The aim of this study was to search for germ-line mutations of the APC gene in unrelated FAP families from southern Spain. By direct sequencing of all APC gene exons, we found the mutation in 13 of 15 unrelated FAP families studied. We identified eight novel mutations: 707delA (exon6), 730_731delAG (exon7), 1787C-->G and 1946_1947insG (exon14), 2496delC, 2838_2839delAT, 2977A-->T, and 3224dupA (exon15). Two patients presented de novo germ-line mutations. Genotype-phenotype correlations for extraintestinal and extracolonic manifestations were studied. Intrafamilial phenotypic variability was observed in two families with mutations in exon/intron boundary, probably due to alternative splicing.     

Regulating DNA Replication in Eukarya

DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication.The Eukarya include a wide spectrum of organisms, with genome sizes ranging from ∼10⁷ bp in yeasts to ∼10¹² bp in protozoa. Rapid duplication of large genomes is achieved by distribution of the genetic material across several chromosomes. Each of these chromosomes initiates replication from sites called replication origins, which must fire no more than once per cell cycle to ensure a single error-free copy of the genome. Generating replication forks from an origin more than once leads to rereplication, an event that creates multiple copies of a single genomic region within a single cell. This leads to gene amplification and promotes genome instability (Green et al. 2010), a phenomenon observed in many human cancers (Lengauer et al. 1998). The process of genome duplication is therefore under stringent control to ensure that few, if any, defects are transmitted from one generation to the next.     

Self-association of the APC tumor suppressor is required for the assembly,stability, and activity of the Wnt signaling destruction complex

The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3β, and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles we termed the destructosome. Whereas APC functions in the complex through its ability to bind both β-cat and Axin, we hypothesize that APC proteins play an additional role in destructosome assembly through self-association. Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.     

Different familial adenomatous polyposis phenotypes resulting from deletions of the entire APC exon 15

Germline mutations of the adenomatous polyposis coli ( APC) gene cause familial adenomatous polyposis (FAP), an autosomal, dominantly inherited disease that predisposes patients to colorectal cancer. The APC gene is composed of 15 coding exons and encodes an open reading frame of 8.5 kb. The 3' 6.5 kb of the APCopen reading frame is encoded by a single exon, exon 15. Most identified APC mutations are at the 5' half of the APC open reading frame and are nucleotide substitutions and small deletions or insertions that result in truncation of the APC protein. Very few well-characterized gross alterations of APC have been reported. Patients with FAP typically develop hundreds to thousands of colorectal tumors beginning in their adolescence. A subgroup of patients with FAP who develop fewer tumors at an older age have what is called attenuated FAP (AFAP). Accumulating evidence indicates that patients carrying germline APC mutations in the first four coding exons, in the alternatively spliced region of exon 9, or in the 3' half of the coding region usually develop AFAP. We characterized two germline APC alterations that deleted the entire APC exon 15 as the result of 56-kb and 73-kb deletions at the APC locus. A surprising finding was that one proband had the typical FAP phenotype, whereas the other had a phenotype consistent with that of AFAP.     

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen – the main component of the anchoring fibrils at the dermal–epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5′ donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.     

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).     

Genome-wide copy number variation analysis of a Branchio-oto-renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus, in total five probands (14 %) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR.     

Aquaporin10 is a pseudogene in cattle and their relatives

BackgroundAlthough AQP10 is mainly expressed in the human GI tract, its physiological role is unclear. In fact, we previously reported that mouse AQP10 is a pseudogene. It is possible that AQP10 is also a pseudogene in other animals.MethodsGenome databases were searched for AQP10 orthologs and the genomic DNA of each candidate pseudogene was sequenced to confirm its mutations. The expression of the AQP10 mRNA was examined by RT-PCR in the small intestine where human AQP10 is highly expressed.ResultsThe genomic database of some mammals had insertions and deletions in the exons of the AQP10 gene, including cattle (Bos taurus), sheep (Ovis aries) and goats (Capra hircus). In the bovine AQP10 gene, exon 1 and 5 had deletions resulting in a frame-shift or a premature termination, respectively, which were confirmed by the direct exon sequencing of the genomic DNA. In the RT-PCR experiments, the PCR primer sets for exon 1/2 and exon 4/5 failed to detect the bands for AQP10 mRNA in the duodenum and jejunum. Similar AQP10 gene mutations were also confirmed in the genomic DNA from sheep and goats. Although these animals were derived from porcine ancestors, the exons of the swine (Sus scrofa) AQP10 gene were complete without mutations. Therefore, AQP10 gene might have turned to a pseudogene around 65 million years before when cattle evolved from porcine ancestors.ConclusionAQP10 of ruminantia which regurgitate and rechew their food may have lost its role possibly due to the redundant expression of other aquaglyceroporins.     

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.