Human rDNA and Cancer

In human cells, each rDNA unit consists of the ~13 kb long ribosomal part and ~30 kb long intergenic spacer (IGS). The ribosomal part, transcribed by RNA polymerase I (pol I), includes genes coding for 18S, 5.8S, and 28S RNAs of the ribosomal particles, as well as their four transcribed spacers. Being highly repetitive, intensively transcribed, and abundantly methylated, rDNA is a very fragile site of the genome, with high risk of instability leading to cancer. Multiple small mutations, considerable expansion or contraction of the rDNA locus, and abnormally enhanced pol I transcription are usual symptoms of transformation. Recently it was found that both IGS and the ribosomal part of the locus contain many functional/potentially functional regions producing non-coding RNAs, which participate in the pol I activity regulation, stress reactions, and development of the malignant phenotype. Thus, there are solid reasons to believe that rDNA locus plays crucial role in carcinogenesis. In this review we discuss the data concerning the human rDNA and its closely associated factors as both targets and drivers of the pathways essential for carcinogenesis. We also examine whether variability in the structure of the locus may be blamed for the malignant transformation. Additionally, we consider the prospects of therapy focused on the activity of rDNA.

WWOX-Related Neurodevelopmental Disorders: Models and Future Perspectives

The WW domain-containing oxidoreductase (WWOX) gene was originally discovered as a putative tumor suppressor spanning the common fragile site FRA16D, but as time has progressed the extent of its pleiotropic function has become apparent. At present, WWOX is a major source of interest in the context of neurological disorders, and more specifically developmental and epileptic encephalopathies (DEEs). This review article aims to introduce the many model systems used through the years to study its function and roles in neuropathies. Similarities and fundamental differences between rodent and human models are discussed. Finally, future perspectives and promising research avenues are suggested.

Mechanisms of Genome Instability in the Fragile X-Related Disorders

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5′ UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54–200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

Plasma exosome-derived fragile-site associated tumor suppressor is a powerful predictor of prognosis in patients with ovarian cancer

Objective: To investigate the levels of plasma exosome-derived fragile-site associated tumor suppressor (FATS) and evaluate its predictive ability in ovarian cancer (OC) patients. Patients and Methods: Exosome-rich fractions were isolated from the plasma of enrolled 90 patients with OC. The levels of plasma exosome-derived FATS were detected with ELISA. Results: The levels of exosome-derived FATS in OC patient were significantly lower than in healthy controls (P < 0.001). The levels of plasma exosome-derived FATS were obviously higher in OC patients with low grade (1/2), FIGO stages I/II than high grade (3/4), stages III/ IV disease (P = 0.003; P < 0.001). The levels of plasma exosome-derived FATS were significantly higher in OC patients with no lymph node metastasis, no ascites than those with lymph node metastasis, ascites (both P < 0.001). The levels of plasma exosome-derived FATS were obviously higher in OC patients with CA-125 less than 35U/ml than more than 35U/ml (P < 0.001). Among all enrolled OC patients, both 5-DFS and 5-OS were shorter in patients who had low plasma exosome-derived FATS levels than that high levels (both P < 0.001). The AUROC of plasma exosome-derived FATS were 0.85(95% CI: 0.76-0.91) for 5-DFS, 0.91(95% CI: 0.83-0.96) for 5-OS prediction in patients with OC, respectively. Conclusions: Plasma exosome-derived FATS levels in OC patient were significantly down-regulated. Low levels of plasma exosome-derived FATS had close relationship with FIGO stages I/II, low grade, ascites, higher levels of CA-125, lymph node metastasis and prognosis of OC patients. Our findings may provide a new strategy in treating OC.

Levodopa-responsive dystonia caused by biallelic PRKN exon inversion invisible to exome sequencing

Biallelic pathogenic variants in PRKN (PARK2), encoding the E3 ubiquitin ligase parkin, lead to early-onset Parkinson's disease. Structural variants, including duplications or deletions, are common in PRKN due to its location within the fragile site FRA6E. These variants are readily detectable by copy number variation analysis. We studied four siblings with levodopa-responsive dystonia by exome sequencing followed by genome sequencing. Affected individuals developed juvenile levodopa-responsive dystonia with subsequent appearance of parkinsonism and motor fluctuations that improved by subthalamic stimulation. Exome sequencing and copy number variation analysis were not diagnostic, yet revealed a shared homozygous block including PRKN. Genome sequencing revealed an inversion within PRKN, with intronic breakpoints flanking exon 5. Breakpoint junction analysis implicated non-homologous end joining and possibly replicative mechanisms as the repair pathways involved. Analysis of cDNA indicated skipping of exon 5 (84 bp) that was replaced by 93 bp of retained intronic sequence, preserving the reading frame yet altering a significant number of residues. Balanced copy number inversions in PRKN are associated with a severe phenotype. Such structural variants, undetected by exome analysis and by copy number variation analysis, should be considered in the relevant clinical setting. These findings raise the possibility that PRKN structural variants are more common than currently estimated.

Large Intronic Deletion of the Fragile Site Gene PRKN Dramatically Lowers Its Fragility Without Impacting Gene Expression

Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.

Loss of fragile site-associated tumor suppressor promotes antitumor immunity via macrophage polarization

Common fragile sites (CFSs) are specific breakage-prone genomic regions and are present frequently in cancer cells. The (E2-independent) E3 ubiquitin-conjugating enzyme FATS (fragile site-associated tumor suppressor) has antitumor activity in cancer cells, but the function of FATS in immune cells is unknown. Here, we report a function of FATS in tumor development via regulation of tumor immunity. Fats−/− mice show reduced subcutaneous B16 melanoma and H7 pancreatic tumor growth compared with WT controls. The reduced tumor growth in Fats−/− mice is macrophage dependent and is associated with a phenotypic shift of macrophages within the tumor from tumor-promoting M2-like to antitumor M1-like macrophages. In addition, FATS deficiency promotes M1 polarization by stimulating and prolonging NF-κB activation by disrupting NF-κB/IκBα negative feedback loops and indirectly enhances both CD4+ T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) adaptive immune responses to promote tumor regression. Notably, transfer of Fats−/− macrophages protects mice against B16 melanoma. Together, these data suggest that FATS functions as an immune regulator and is a potential target in cancer immunotherapy.

Non-canonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast

Long-tract gene conversions (LTGC) can result from the repair of collapsed replication forks, and several mechanisms have been proposed to explain how the repair process produces this outcome. We studied LTGC events produced from repair collapsed forks at yeast fragile site FS2. Our analysis included chromosome sizing by contour-clamped homogeneous electric field (CHEF) electrophoresis, next-generation whole genome sequencing, and Sanger sequencing across repair event junctions. We compared the sequence and structure of LTGC events in our cells to the expected qualities of LTGC events generated by proposed mechanisms. Our evidence indicates that some LTGC events arise from half-crossover during BIR, some LTGC events arise from gap repair, and some LTGC events can be explained by either gap repair or “late” template switch during BIR. Also based on our data, we propose that models of collapsed replication forks be revised to show not a one-end double-strand break (DSB), but rather a two-end DSB in which the ends are separated in time and subject to gap repair.

Molecular Biology of the WWOX Gene That Spans Chromosomal Fragile Site FRA16D

It is now more than 20 years since the FRA16D common chromosomal fragile site was characterised and the WWOX gene spanning this site was identified. In this time, much information has been discovered about its contribution to disease; however, the normal biological role of WWOX is not yet clear. Experiments leading to the identification of the WWOX gene are recounted, revealing enigmatic relationships between the fragile site, its gene and the encoded protein. We also highlight research mainly using the genetically tractable model organism Drosophila melanogaster that has shed light on the integral role of WWOX in metabolism. In addition to this role, there are some particularly outstanding questions that remain regarding WWOX, its gene and its chromosomal location. This review, therefore, also aims to highlight two unanswered questions. Firstly, what is the biological relationship between the WWOX gene and the FRA16D common chromosomal fragile site that is located within one of its very large introns? Secondly, what is the actual substrate and product of the WWOX enzyme activity? It is likely that understanding the normal role of WWOX and its relationship to chromosomal fragility are necessary in order to understand how the perturbation of these normal roles results in disease.

Plasma Exosome-Derived Fragile-Site Associated Tumor Suppressor is a Powerful Predictor of Prognosis in Patients with Ovarian Cancer

Objective: To investigate the levels of plasma exosome-derived fragile-site associated tumor suppressor (FATS) and evaluate its predictive ability in ovarian cancer (OC) patients.Patients and Methods: Exosome-rich fractions were isolated from the plasma of enrolled 90 patients with OC. The levels of plasma exosome-derived FATS were detected with ELISA.Results: The levels of exosome-derived FATS in OC patient were significantly lower than in healthy controls (P < 0.001). The levels of plasma exosome-derived FATS were obviously higher in OC patients with low grade (1/2), FIGO stages I/II than high grade (3/4), stages III/ IV disease (P = 0.003; P < 0.001). The levels of plasma exosome-derived FATS were significantly higher in OC patients with no lymph node metastasis, no ascites than those with lymph node metastasis, ascites (both P < 0.001). The levels of plasma exosome-derived FATS were obviously higher in OC patients with CA-125 less than 35U/ml than more than 35U/ml (P < 0.001). Among all enrolled OC patients, both 5-DFS and 5-OS were shorter in patients who had low plasma exosome-derived FATS levels than that high levels (both P < 0.001). The AUROC of plasma exosome-derived FATS were 0.85(95% CI: 0.76-0.91) for 5-DFS, 0.91(95% CI: 0.83-0.96) for 5-OS prediction in patients with OC, respectively.Conclusions: Plasma exosome-derived FATS levels in OC patient were significantly down-regulated. Low levels of plasma exosome-derived FATS had close relationship with FIGO stages I/II, low grade, ascites, higher levels of CA-125, lymph node metastasis and prognosis of OC patients. Our findings may provide a new strategy in treating OC.