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.

     

Mutational analysis of patients with adenomatous polyposis: Identical inactivating mutations in unrelated individuals

Samples of constitutional DNA from 60 unrelated patients with adenomatous polyposis coli (APC) were examined for mutations in the APC gene. Five inactivating mutations were observed among 12 individuals with APC; all were different from the six inactivating mutations previously reported in this panel of patients. The newly discovered mutations included single-nucleotide substitutions leading to stop codons and small deletions leading to frameshifts. Two of the mutations were observed in multiple APC families and in sporadic cases of APC; allele-specific PCR primers were designed for detecting mutations at these common sites. No missense mutations that segregated with the disease were found.     

Aneuploid colon cancer cells have a robust spindle checkpoint

Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused by mismatch repair defects, the lesions responsible for CIN are unknown. The observation that CIN cells fail to undergo mitotic arrest following spindle damage suggested that mutations in spindle checkpoint genes may account for CIN. However, here we show that CIN cells do undergo mitotic arrest in response to spindle damage. Although the maximum mitotic index achieved by CIN lines is diminished relative to MIN lines, CIN cells clearly have a robust spindle checkpoint. Consistently, mutations in spindle checkpoint genes are rare in human tumours. In contrast, the adenomatous polyposis coli (APC) gene is frequently mutated in CIN cells. Significantly, we show here that expression of an APC mutant in MIN cells reduces the mitotic index following spindle damage to a level observed in CIN cells, suggesting that APC dysfunction may contribute to CIN.     

Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress

DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high‐throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin‐treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C‐terminal dual serine target motif unique to H2AX in the plant lineage showed 171‐fold phosphorylation that was absent in atm mutant lines. The physiological significance of post‐translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM‐mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post‐translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress.     

Initiation of DNA damage responses through XPG‐related nucleases

Lesion‐specific enzymes repair different forms of DNA damage, yet all lesions elicit the same checkpoint response. The common intermediate required to mount a checkpoint response is thought to be single‐stranded DNA (ssDNA), coated by replication protein A (RPA) and containing a primer‐template junction. To identify factors important for initiating the checkpoint response, we screened for genes that, when overexpressed, could amplify a checkpoint signal to a weak allele of chk1 in fission yeast. We identified Ast1, a novel member of the XPG‐related family of endo/exonucleases. Ast1 promotes checkpoint activation caused by the absence of the other XPG‐related nucleases, Exo1 and Rad2, the homologue of Fen1. Each nuclease is recruited to DSBs, and promotes the formation of ssDNA for checkpoint activation and recombinational repair. For Rad2 and Exo1, this is independent of their S‐phase role in Okazaki fragment processing. This XPG‐related pathway is distinct from MRN‐dependent responses, and each enzyme is critical for damage resistance in MRN mutants. Thus, multiple nucleases collaborate to initiate DNA damage responses, highlighting the importance of these responses to cellular fitness.     

The value of MUTYH testing in patients with early onset microsatellite stable colorectal cancer referred for hereditary nonpolyposis colon cancer syndrome testing

MUTYH adenomatous polyposis (MAP) can mimic both the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) phenotypes. As a result of MAP's phenotypic overlap with FAP, some DNA diagnostic laboratories perform MUTYH testing in conjunction with APC testing in patients with suspected FAP or attenuated FAP (AFAP). In addition to testing FAP/AFAP samples for MUTYH mutations, we were interested whether there would also be value in testing samples referred for HNPCC testing. To determine this, we tested a consecutive series of 229 samples referred for HNPCC testing for the two most common MUTYH mutations in the Caucasian population. To enrich our study population with MAP cases, we only included samples from patients with early onset colorectal cancer (CRC diagnosed <50 years old) in whom HNPCC had been excluded by microsatellite instability testing (microsatellite stable or low microsatellite instability). Four biallelic (2%) and six monoallelic (3%) MUTYH mutation carriers were identified. No clinical factors predicted MUTYH mutation status. Specifically, a family history of vertical transmission of CRC or having few polyps (<15) did not rule out the possibility of biallelic MUTYH mutations. Thus, MUTYH mutation testing may be a reasonable cascade test in early onset CRC found to have proficient DNA mismatch repair, regardless of pattern of family history or number of polyps.     

Frequent polymorphism in the 13th exon of the adenomatous polyposis coli gene

Summary We have studied the DNA from 170 individuals affected with familial adenomatous polyposis and from 20 uneffected individuals. Denaturing gradient gel electrophoresis of polymerase chain reaction amplified DNA from the adenomatous polyposis coli (APC) gene demonstrated three major patterns consistent with the existence of two different sequences in exon 13 of the gene in the human population. Direct sequencing of the amplified product from DNAs producing these three patterns confirmed the presence of a polymorphism in the coding region of the APC gene.     

Altered Interactions between the Gut Microbiome and Colonic Mucosa Precede Polyposis in APCMin/+ Mice

Mutation of the adenomatous polyposis coli (APC gene), an early event in the adenoma-carcinoma sequence, is present in 70-80% of sporadic human colorectal adenomas and carcinomas. To test the hypothesis that mutation of the APC gene alters microbial interactions with host intestinal mucosa prior to the development of polyposis, culture-independent methods (targeted qPCR assays and Illumina sequencing of the 16S rRNA gene V1V2 hypervariable region) were used to compare the intestinal microbial composition of 30 six-week old C57BL/6 APC^Min/+ and 30 congenic wild type (WT) mice. The results demonstrate that similar to 12-14 week old APC^Min/+ mice with intestinal neoplasia, 6 week old APC^Min/+ mice with no detectable neoplasia, exhibit an increased relative abundance of Bacteroidetes spp in the colon. Parallel mouse RNA sequence analysis, conducted on a subset of proximal colonic RNA samples (6 APC^Min/+, 6 WT) revealed 130 differentially expressed genes (DEGs, fold change ≥ 2, FDR <0.05). Hierarchical clustering of the DEGs was carried out by using 1-r dissimilarity measurement, where r stands for the Pearson correlation, and Ward minimum variance linkage, in order to reduce the number of input variables. When the cluster centroids (medians) were included along with APC genotype as input variables in a negative binomial (NB) regression model, four of seven mouse gene clusters, in addition to APC genotype, were significantly associated with the increased relative abundance of Bacteroidetes spp. Three of the four clusters include several downregulated genes encoding immunoglobulin variable regions and non-protein coding RNAs. These results support the concept that mutation of the APC gene alters colonic-microbial interactions prior to polyposis. It remains to be determined whether interventions directed at ameliorating dysbiosis in APC^Min/+mice, such as through probiotics, prebiotics or antibiotics, could reduce tumor formation.     

APC shuttling to the membrane, nucleus and beyond

The adenomatous polyposis coli (APC) tumor suppressor is a multi-functional protein, the mutation of which triggers colon cancer progression through de-regulation of the canonical Wnt signaling pathway and disruption of the mitotic spindle checkpoint. In recent years, APC has been detected at several unexpected intracellular locations, implicating APC in multiple roles that now include the regulation of directed cell migration, apoptosis and DNA repair. In this review, we discuss the intracellular trafficking pathway of APC and describe how truncated cancer-mutant forms of APC display frequent changes in sub-cellular localization and function. The transport routes of APC overlap that of other tumor suppressors, including BRCA1 and p53, pin-pointing common destinations and functions for these cancer regulators.     

γH2A binds Brc1 to maintain genome integrity during S‐phase

ATM^Tel1 and ATR^Rad3 checkpoint kinases phosphorylate the C‐terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho‐H2A/X (γH2A/X)‐binding proteins at double‐strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X‐ray scattering (SAXS), and X‐ray structural studies of the Schizosaccharomyces pombe Brc1, a 6‐BRCT‐domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds γH2A to form spontaneous and DNA damage‐induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage‐induced Brc1 foci colocalize with DSB response factors. γH2A binding is critical for Brc1 function. The 1.45 Å resolution crystal structure of Brc1–γH2A complex shows how variable BRCT insertion loops sculpt tandem‐BRCT phosphoprotein‐binding pockets to facilitate unique phosphoprotein‐interaction specificities, and unveils an acidic DNA‐mimicking Brc1 surface. From these results, Brc1 docking to γH2A emerges as a critical chromatin‐specific response to replication‐associated DNA damage.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

DNA repair and cell cycle checkpoint defects as drivers and therapeutic targets in melanoma

The ultraviolet radiation (UVR) component of sunlight is the major environmental risk factor for melanoma, producing DNA lesions that can be mutagenic if not repaired. The high level of mutations in melanomas that have the signature of UVR‐induced damage indicates that the normal mechanisms that detect and repair this damage must be defective in this system. With the exception of melanoma‐prone heritable syndromes which have mutations of repair genes, there is little evidence for somatic mutation of known repair genes. Cell cycle checkpoint controls are tightly associated with repair mechanisms, arresting cells to allow for repair before continuing through the cell cycle. Checkpoint signaling components also regulate the repair mechanisms. Defects in checkpoint mechanisms have been identified in melanomas and are likely to be responsible for increased mutation load in melanoma. Loss of the checkpoint responses may also provide an opportunity to target melanomas using a synthetic lethal approach to identify and inhibit mechanisms that compensate for the defective checkpoints.     

Chronic exposure to the cytolethal distending toxins of Gram‐negative bacteria promotes genomic instability and altered DNA damage response

Epidemiological evidence links chronic bacterial infections to the increased incidence of certain types of cancer but the molecular mechanisms by which bacteria contribute to tumour initiation and progression are still poorly characterized. Here we show that chronic exposure to the genotoxin cytolethal distending toxin (CDT) of Gram‐negative bacteria promotes genomic instability and acquisition of phenotypic properties of malignancy in fibroblasts and colon epithelial cells. Cells grown for more than 30 weeks in the presence of sublethal doses of CDT showed increased mutation frequency, and accumulation of chromatin and chromosomal aberrations in the absence of significant alterations of cell cycle distribution, decreased viability or senescence. Cell survival was dependent on sustained activity of the p38 MAP kinase. The ongoing genomic instability was associated with impaired activation of the DNA damage response and failure to efficiently activate cell cycle checkpoints upon exposure to genotoxic stress. Independently selected sublines showedenhanced anchorage‐independent growth as assessed by the formation of colonies in semisolid agarose. These findings support the notion that chronic infection by CDT‐producing bacteria may promote malignant transformation, and point to the impairment of cellular control mechanisms associated with the detection and repair of DNA damage as critical events in the process.     

Structure/function analysis of the interaction of adenomatous polyposis coli with DNA polymerase beta and its implications for base excision repair

Mutations in the adenomatous polyposis coli (APC) gene are associated with an early onset of colorectal carcinogenesis. Previously, we described a novel role for the APC polypeptide in base excision repair (BER). The single-nucleotide (SN) and long-patch (LP) BER pathways act to repair the abasic sites in DNA that are induced by stressors, such as spontaneous oxidation/reduction, alkylation, and hyperthermia. We have shown that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 (Fen-1) and blocks Pol-beta-directed strand-displacement synthesis. In this study, we have mapped the APC interaction site in Pol-beta and have found that Thr79, Lys81, and Arg83 of Pol-beta were critical for its interaction with APC. The Pol-beta protein (T79A/K81A/R83A) blocked strand-displacement DNA synthesis in which tetrahydrofuran was used as DNA substrate. We further showed that the APC-mediated blockage of LP-BER was due to inhibition of Fen-1 activity. Analysis of the APC-mediated blockage of SN-BER indicated that the interaction of APC with Pol-beta blocked SN-BER activity by inhibiting Pol-beta-directed deoxyribose phosphate lyase activity. Collectively, our findings indicate that APC blocked both Pol-beta-directed SN- and LP-BER pathways and increased sensitivity of cells to alkylation induced DNA damage.     

Studying the DNA damage response using in vitro model systems

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order to prevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathways that delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis of ATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro model systems to understand the detailed mechanisms underlying their function. Here we describe some recent findings obtained analysing these pathways using in vitro model systems. In particular we focus on the studies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response in the context of the cell cycle.     

DNA‐PK suppresses a p53‐independent apoptotic response to DNA damage

p53 is required for DNA damage‐induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53‐deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA‐dependent protein kinase (DNA‐PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation‐induced apoptosis, whereas apoptosis in DNA‐PK_cs/p53, Ku80/p53 and Ku70/p53 double‐null mice was quantitatively equivalent to that seen in wild‐type mice. This p53‐independent apoptotic response was specific to the loss of DNA‐PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double‐null mice. Furthermore, it was associated with an increase in phospho‐checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA‐PK, does not require p53.