FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites

The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes for the formation of gaps and breaks when DNA replication is perturbed by aphidicolin or folate stress. Although rare fragile sites are known to arise through the expansion of CCG repeats, the mechanism responsible for common fragile sites is unknown. Beyond being a basic component of chromosome structure, no biological effects of common fragile sites have been convincingly shown, although suggestions have been made that breakage and recombination at these sites may sometimes be mechanistically involved in deletions observed in many tumors and in constitutional deletions. In an observation related to the high rate of recombination at fragile sites, a number of studies have shown a statistical association between the integration of transforming DNA viruses and chromosomal fragile sites. Using FISH analysis we recently identified a 1.3 Mb YAC spanning both FRA3B and the t(3;8) translocation associated with hereditary RCC. Here we report the further localization of FRA3B within this YAC. Using lambda subclones of the YAC as FISH probes, gaps and breaks were found to occur over a broad region of at least 50 kb. Neither CCG nor CAG repeats were found in this region suggesting a different mechanism for fragility than seen with rare fragile sites. We further show that an area of frequent gaps and breaks within FRA3B, defined by a lambda contig, coincides with a previously characterized site of HPV16 integration in a primary cervical carcinoma. The HPV16 integration event gave rise to a short chromosomal deletion limited to the local FRA3B region within 3p14.2. Interestingly, 3p14.2 lies within the smallest commonly deleted region of 3p in cervical cancers, which are often HPV16 associated. To our knowledge this is the first molecular characterization of an in vivo viral integration event within a confirmed fragile site region, supporting previous cytogenetic observations linking viral integration sites and fragile sites.      

Integrated YAC contig containing the 3pl4.2 hereditary renal carcinoma 3;8 translocation breakpoint and the fragile site FRA3B

An extended YAC contig has been developed for the 3p14 region containing the hereditary renal carcinoma 3;8 translocation breakpoint and the 3p 14.2 fragile site FRA3B. This region of chromosome 3 has been implicated by chromosomal translocation, deletion, and loss of heterozygosity in the pathogenesis of several malignant diseases. The contig allows accurate positioning of candidate genes, polymorphic markers, and other 3p rearrangements within this region. The contig, spanning approximately 6 Mb of DNA, contains 51 YACs identified by 27 markers, including a subset of CA repeats located in the 3p 14.1–14.2 interval. The order of CA microsatellites, derived from marker content of the YACs, is in agreement with the order previously determined by genetic linkage studies. We find that the protein-tyrosine phosphatase gamma gene, PTPRG, is located minimally 1 Mb proximal to the t(3;8) breakpoint The more proximal 3p homozygous deletion in the small-cell lung cancer cell line, U2020, is more than 5 Mb from the site of the 3;8 translocation. This integrated physical and genetic map provides a framework for further investigations of malignant diseases associated with proximal 3p loss. In addition, the positioning of separate 3p 14.2 aphidicolin-induced breakpoints suggests that FRA3B may represent a region rather than a single site.     

Mechanisms shaping the mutational landscape of the FRA3B/FHIT‐deficient cancer genome

Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base‐pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT‐loss causes replication stress, DNA breaks, aneuploidy, copy‐number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT‐loss‐induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single‐stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT‐deficiency is highly associated with the ubiquitous, clock‐like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT‐deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.     

FHIT gene in gastric cancer: association with tumour progression and prognosis

The FHIT (fragile histidine triad) gene has been recently identified and cloned at chromosome 3p14.2 including FRA3B, the most common fragile site in the human genome. FHIT is suggested to be a candidate tumour suppressor gene in gastrointestinal tract tumours. To elucidate the role of the FHIT gene in gastric cancer, a total of 133 curatively R0-resected gastric carcinomas were investigated for loss of heterozygosity (LOH) at 3p14.2, using four polymorphic microsatellite loci (D3S1300, D3S1313, D3S1481, and D3S1234). LOH of the FHIT gene affecting at least one of the investigated loci was observed in 20 of 123 informative tumours (16·3 per cent). The presence of LOH was correlated neither with major prognostic factors such as pT category, pN category or vascular invasion, nor with histological type or grade of differentiation of the tumours. In addition, there were no differences in the prognosis between patients with gastric carcinomas showing LOH at the FHIT gene and patients with tumours lacking LOH at the FHIT gene. These findings suggest that LOH of the FHIT gene represents an event in the tumourigenesis of only a small subset of gastric carcinomas and does not correlate with tumour progression or prognosis. Copyright © 1999 John Wiley & Sons, Ltd.     

Common chromosome fragile sites in human and murine epithelial cells and FHIT/FRA3B loss‐induced global genome instability

Chromosomal positions of common fragile sites differ in lymphoblasts and fibroblasts, with positions dependent on the epigenetically determined density of replication origins at these loci. Because rearrangement of fragile loci and associated loss of fragile gene products are hallmarks of cancers, we aimed to map common fragile sites in epithelial cells, from which most cancers derive. Among the five most frequently activated sites in human epithelial cells were chromosome bands 2q33 and Xq22.1, which are not among top fragile sites identified in lymphoblasts or fibroblasts. FRA16D at 16q23 was among the top three fragile sites in the human epithelial cells examined, as it is in lymphoblasts and fibroblasts, while FRA3B at 3p14.2, the top fragile locus in lymphoblasts, was not fragile in most epithelial cell lines tested. Epithelial cells exhibited varying hierarchies of fragile sites; some frequent epithelial cell fragile sites are apparently not frequently altered in epithelial cancers and sites that are frequently deleted in epithelial cancers are not necessarily among the most fragile. Since we have reported that loss of expression of the FRA3B‐encoded FHIT protein causes increased replication stress‐induced DNA damage, we also examined the effect of FHIT‐deficiency on markers of genome instability in epithelial cells. FHIT‐deficient cells exhibited increases in fragile breaks and in γH2AX and 53BP1 foci in G1 phase cells, confirming in epithelial cells that the FHIT gene and encompassing FRA3B, is a “caretaker gene” necessary for maintenance of genome stability. © 2013 Wiley Periodicals, Inc.     

Genetic instability of the tumor suppressor gene FHIT in normal human cells

Common fragile sites are hotspots for chromosome instability and co‐localize to cancer genomic rearrangements. Whether these loci may be considered stable in human subjects under physiological conditions remains an open question. Here we show by molecular combing that a small but significant percentage of normal human cells carry an abnormal sequence pattern within the tumor suppressor gene FHIT (3p14.2) at FRA3B. Each sequence variation represents a unique pattern within a normal cell population, and therefore it would remain undetected or not interpreted by genome‐wide analyses. Remarkably, the region is the same as in FHIT rearrangements described in tumors. By analyses on several normal cell lines (proliferating and resting primary lymphocytes, primary fibroblasts, lymphoblastoid cells including clonal cell cultures) we verified that: (a) each cell type displays altered sequence patterns at FHIT; (b) the presence of abnormal sequence patterns is specific for the FHIT locus; and (c) FHIT instability occurs de novo during cell proliferation, and heterogeneous sequence variants progressively accumulate in the cell populations. FHIT has been widely investigated in cancer cells, but to our knowledge this is the first direct evidence of spontaneous and recurrent occurrence of genomic instability at this gene in human subjects, at the same region involved in cancer rearrangements. Our results suggest that common fragile site activity is not restricted to in vitro cell culture and that genomic instability may pre‐exist in normal cells in the absence of exogenous replication stress. © 2013 Wiley Periodicals, Inc.     

Allele-specific late replication and fragility of the most active common fragile site, FRA3B

FRA3B at 3p14.2 is the most active of the common fragile sites in the human genome and is expressed when cells are exposed to the DNA replication inhibitor, aphidicolin. Several lines of evidence suggest that fragile sites are regions of late replication. To elucidate the relationship between the timing of replication across the FRA3B region and its corresponding fragility, we labeled cells with 5-bromo-2'- deoxyuridine (BrdU) and adopted an immunofluorescent procedure to visualize late replicating DNA (BrdU-substituted DNA) in metaphase chromosomes. We also chose 21 markers along the FRA3B region and analyzed the timing of replication using BrdU-labeled DNA from different stages of the cell cycle sorted by flow cytometry. Our results show that there are two distinct alleles that replicate at different stages in the cell cycle and that breaks/gaps preferentially occurred on the chromosome 3 with the late replication allele. These results provide direct evidence that allele-specific late replication is involved in the fragility of the most active common fragile site, FRA3B.      

The FRA14B common fragile site maps to a region prone to somatic and germline rearrangements within the large GPHN gene

Common fragile sites (cFSs) represent parts of the normal chromosome structure susceptible to breakage under replication stress. Although only a small number of cFSs have been molecularly characterized, genomic damage of cFS genes appears to be critical for the development of various human diseases. In this study, we fine mapped the location of FRA14B and showed that the fragile region spans 765 kb at 14q23.3, containing the large gephyrin (GPHN) gene. The FRA14B sequence is enriched in perfect A/T_>24 stretches and R‐loop forming sequences (RLFS), and harbors a large palindromic motif in the core region. FRA14B instability is not only limited to lymphocytes, but also occurs in neuroblastoma and breast epithelial cells. Using array comparative genomic hybridization (CGH), we examined copy number alteration patterns within FRA14B in a panel of 180 cancer cell lines and primary tumors. Our CGH data and a survey of 1046 Cancer Cell Line Encyclopedia profiles demonstrate that focal deletions cluster within FRA14B and disrupt the genomic integrity of GPHN in approximately 5% of cancer cells. Moreover, germline CNVs (copy number variants) profiles provided by the Database of Genomic Variants and available literature suggest that germline CNVs and rare pathogenic deletions associated with neurodevelopmental disorders cluster within the core fragile region of GPHN. Overall, our data provide insight into the molecular structure of FRA14B, and identify GPHN, as a large cFS gene in the human genome, whose disruption appears to trigger various neurodevelopmental diseases.     

Analysis of replication timing at the FRA10B and FRA16B fragile site loci

The molecular basis for the cytogenetic appearance of chromosomal fragile sites is not yet understood. Late replication and further delay of replication at fragile sites expressing alleles has been observed for FRAXA, FRAXE and FRA3B fragile site loci. We analysed the timing of replication at the FRA10B and FRA16B loci to determine whether late replication is a feature which is shared by all fragile sites and, therefore, is a necessary condition for chromosomal fragile site expression. The FRA10B locus was located in a transitional region between early and late zones of replication. Fragile and non-fragile alleles exhibit a similar replication pattern proximal to the repeat, but fragile alleles are delayed relative to non-fragile ones on the distal side. Although fragility at FRA10B appears to be caused by expansion of an AT-rich repeat in the region, replication time near the repeat was similar in fragile and non-fragile alleles. The FRA16B locus was late replicating and appeared to replicate even later on fragile chromosomes. While these observations are compatible with the hypothesis that delayed replication may play a role in fragile site expression, they suggest that replication delay may not need to occur at the expanded repeat region itself in order to be permissive for fragility. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal fragile sites FRA3B and FRA16D show correlated expression and association with failure of apoptosis in lymphocytes from patients with thyroid cancer

It has been suggested that common fragile sites (cFSs) are related to cancer development. This appears to be the case for FRA3B and FRA16D, localized in two tumor-suppressor genes (FHIT and WWOX, respectively) that are altered by deletions or loss of heterozygosity (LOH) in many cancers. The features responsible for fragility have not yet been identified. Furthermore, it is still unclear whether instability at these regions causes chance deletions and loss of function of the associated genes, or whether the gene function itself is related to the appearance of fragility. In this study, we analyzed cFS expression in lymphocytes from 20 healthy or thyroid cancer-affected subjects exposed to radiation after the Chernobyl accident. The same cells were examined for apoptosis, a principal function of both the FHIT and WWOX genes. Exceptionally elevated chromosome fragility was observed, particularly in cancer patients, affecting FRA3B, FRA16D, and a cluster of less highly expressed cFSs; levels of chromosome fragility were found to be correlated among these cFSs. Interestingly, most expressed cFSs were sites of LOH reported for thyroid tumors; moreover, cells with the highest fragility also had a reduced ability to undergo apoptosis. These findings reveal previously unknown genetic interactions affecting fragile loci, suggestive of a shared function inside mitotic cells. Attenuation of checkpoint control and apoptosis resistance seem to be the cell phenotypes associated with unusual chromosome fragility. We propose that breakage at specific cFS could derive from early epigenetic events at loci involved in radiation carcinogenesis. This article contains supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Fish mapping of YAC clones at human chromosomal band 7q31.2: Identification of YACS spanning FRA7G within the common region of LOH in breast and prostate cancer

Loss of DNA sequences within human chromosomal band 7q31.2 is frequently observed in a number of different solid tumors including breast, prostate, and ovarian cancer. This chromosomal band also contains the common fragile site, FRA7G. Many of the common fragile sites occur within chromosomal regions that are frequently deleted during tumor formation but their precise position, relative to the chromosome breakpoints and deletions, has not been defined for the majority of the fragile sites. Because the frequency of expression of FRA7G is low, we analyzed the expression of FRA7G in a chromosome 7-only somatic cell hybrid (hamster-human). YAC clones defining a contig spanning 7q31.2 were then used as FISH probes against metaphase spreads prepared from the hybrid cells after aphidicolin induction. This analysis quickly revealed whether a specific YAC clone mapped proximal, distal, or actually spanned the region of decondensation/breakage of FRA7G. By using this approach, we have identified several overlapping YAC clones that clearly span FRA7G. Interestingly, these clones map precisely to the common region of LOH in breast cancer and prostate cancer. In addition, the MET oncogene is contained within the three YACs that span FRA7G. Genes Chromosomes Cancer 21:152–159, 1998. © 1998 Wiley-Liss, Inc.     

Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction

The FRA3B at 3p14.2 is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin or folate stress. The molecular basis for chromosome fragility at FRA3B is unknown. In contrast to the rare fragile sites, including FRAXA, no repeat motifs, such as trinucleotide repeats, have been identified within FRA3B. Several lines of evidence suggest that fragile sites are regions of DNA whose replication is unusually sensitive to interference. We have used fluorescence in situ hybridization to determine the relative timing of replication of FRA3B sequences. Our studies revealed that FRA3B sequences are late replicating. Exposure to aphidicolin, an inhibitor of both DNA polymerase alpha and delta, results in a reproducible delay in the timing of replication, and some cells enter G2without having completed replication of FRA3B sequences. Our results support a model in which common fragile sites are sequences that initiate replication late in S phase or are slow to replicate, and the chromosomal breaks and gaps observed in metaphase cells are due to unreplicated DNA.