Large-scale Genomic Alterations Research Articles

Genomic structural variations link multiple genes to bone mineral density in a multi-ethnic cohort study: Louisiana osteoporosis study.

Osteoporosis, characterized by low bone mineral density (BMD), is a highly heritable metabolic bone disorder. While single nucleotide variations (SNVs) have been extensively studied, they explain only a fraction of BMD heritability. While genomic structural variations (SVs) are large-scale genomic alterations that contribute to genetic diversity in shaping phenotypic variations, the role of SVs in osteoporosis susceptibility remains poorly understood. This study aims to identify and prioritize genes that harbor BMD-related SVs. We performed whole genome sequencing on 4982 subjects from the Louisiana Osteoporosis Study. To obtain high-confidence SVs, the detection of SVs was performed using an ensemble approach. The SVs were tested for association with BMD variation at the hip (HIP), femoral neck (FNK), and lumbar spine (SPN), respectively. Additionally, we conducted co-occurrence analysis using multi-omics approaches to prioritize the identified genes based on their functional importance. Stratification was employed to explore the sex- and ethnicity-specific effects. We identified significant SV-BMD associations: 125 for FNK-BMD, 99 for SPN-BMD, and 83 for HIP-BMD. We observed SVs that were commonly associated with both FNK and HIP BMDs in our combined and stratified analyses. These SVs explain 13.3% to 19.1% of BMD variation. Novel bone-related genes emerged, including LINC02370, ZNF family genes, and ZDHHC family genes. Additionally, FMN2, carrying BMD-related deletions, showed associations with FNK or HIP BMDs, with sex-specific effects. The co-occurrence analysis prioritized an RNA gene LINC00494 and ZNF family genes positively associated with BMDs at different skeletal sites. Two potential causal genes, IBSP and SPP1, for osteoporosis were also identified. Our study uncovers new insights into genetic factors influencing BMD through SV analysis. We highlight BMD-related SVs, revealing a mix of shared and specific genetic influences across skeletal sites and gender or ethnicity. These findings suggest potential roles in osteoporosis pathophysiology, opening avenues for further research and therapeutic targets.

Candida albicans' inorganic phosphate transport and evolutionary adaptation to phosphate scarcity.

Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling, oxidative stress resistance and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. Initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity eventually resolved, as populations gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while severity of others like membrane stress responses diverged from Pi scarcity fitness. Among preliminary candidate genes for contributors to fitness recovery, those with links to TORC1 were overrepresented. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth, stress resistance and virulence.

Cell-free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer.

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour-enriched tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next-generation sequencing was performed on ascites-derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites-derived tumour cells (n = 5) and archived formalin-fixed paraffin-embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

The Genomic Shock Hypothesis: Genetic and Epigenetic Alterations of Transposable Elements after Interspecific Hybridization in Plants.

Transposable elements (TEs) are major components of plant genomes with the ability to change their position in the genome or to create new copies of themselves in other positions in the genome. These can cause gene disruption and large-scale genomic alterations, including inversions, deletions, and duplications. Host organisms have evolved a set of mechanisms to suppress TE activity and counter the threat that they pose to genome integrity. These includes the epigenetic silencing of TEs mediated by a process of RNA-directed DNA methylation (RdDM). In most cases, the silencing machinery is very efficient for the vast majority of TEs. However, there are specific circumstances in which TEs can evade such silencing mechanisms, for example, a variety of biotic and abiotic stresses or in vitro culture. Hybridization is also proposed as an inductor of TE proliferation. In fact, the discoverer of the transposons, Barbara McClintock, first hypothesized that interspecific hybridization provides a "genomic shock" that inhibits the TE control mechanisms leading to the mobilization of TEs. However, the studies carried out on this topic have yielded diverse results, showing in some cases a total absence of mobilization or being limited to only some TE families. Here, we review the current knowledge about the impact of interspecific hybridization on TEs in plants and the possible implications of changes in the epigenetic mechanisms.

Whole genome sequencing for metastatic mutational burden in extraskeletal myxoid chondrosarcoma.

Extraskeletal myxoid chondrosarcoma (EMC) is an ultra-rare cancer that makes up less than 3% of all soft tissue sarcomas. It most often arises in the soft tissues of the proximal limbs and has a higher incidence in males. Though EMC has a good prognosis, it has an indolent course with high rates of local recurrence as well as metastasis to the lungs. EMC is characterized in 70% of cases by an EWS1-NR4A3 translocation, leading to constitutive expression of NR4A3. Structural variants (SVs) in EMC, especially large-scale genomic alterations, have not been well studied and studies are severely limited by sample size. In this study, we describe Whole Genome Sequencing (WGS) of a rare case of matched EMC primary tumor, lung metastasis, and pelvic metastasis to identify genomic alterations. We examined somatic variants, copy number variants (CNVs), and larger scale SVs such as translocations and breakend points. While the primary tumor and lung metastasis had similar somatic variations and CNVs, the pelvic metastasis had more unique SVs with especially increased mutational burden of SVs in chromosome 2. This suggests that different molecular drivers appear in more advanced, relapsing EMC compared with the primary tumor and early lung metastasis. Genomic studies such as ours may identify novel molecular complexities in rare cancers that may be leveraged for therapeutic strategies and precision medicine.

APOBEC mutagenesis, kataegis, chromothripsis in EGFR-mutant osimertinib-resistant lung adenocarcinomas

Studies of targeted therapy resistance in lung cancer have primarily focused on single-gene alterations. Based on prior work implicating apolipoprotein b mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) mutagenesis in histological transformation of epidermal growth factor receptor (EGFR)-mutant lung cancers, we hypothesized that mutational signature analysis may help elucidate acquired resistance to targeted therapies. APOBEC mutational signatures derived from an Food and Drug Administration-cleared multigene panel [Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT)] using the Signature Multivariate Analysis (SigMA) algorithm were validated against the gold standard of mutational signatures derived from whole-exome sequencing. Mutational signatures were decomposed in 3276 unique lung adenocarcinomas (LUADs), including 93 paired osimertinib-naïve and -resistant EGFR-mutant tumors. Associations between APOBEC and mechanisms of resistance to osimertinib were investigated. Whole-genome sequencing was carried out on available EGFR-mutant lung cancer samples (10 paired, 17 unpaired) to investigate large-scale genomic alterations potentially contributing to osimertinib resistance. APOBEC mutational signatures were more frequent in receptor tyrosine kinase (RTK)-driven lung cancers (EGFR, ALK, RET, and ROS1; 25%) compared to LUADs at large (20%, P < 0.001); across all subtypes, APOBEC mutational signatures were enriched in subclonal mutations (P < 0.001). In EGFR-mutant lung cancers, osimertinib-resistant samples more frequently displayed an APOBEC-dominant mutational signature compared to osimertinib-naïve samples (28% versus 14%, P= 0.03). Specifically, mutations detected in osimertinib-resistant tumors but not in pre-treatment samples significantly more frequently displayed an APOBEC-dominant mutational signature (44% versus 23%, P < 0.001). EGFR-mutant samples with APOBEC-dominant signatures had enrichment of large-scale genomic rearrangements (P= 0.01) and kataegis (P= 0.03) in areas of APOBEC mutagenesis. APOBEC mutational signatures are frequent in RTK-driven LUADs and increase under the selective pressure of osimertinib in EGFR-mutant lung cancer. APOBEC mutational signature enrichment in subclonal mutations, private mutations acquired after osimertinib treatment, and areas of large-scale genomic rearrangements highlights a potentially fundamental role for APOBEC mutagenesis in the development of resistance to targeted therapies, which may be potentially exploited to overcome such resistance.

Role of Transposable Elements in Genome Stability: Implications for Health and Disease.

Most living organisms have in their genome a sizable proportion of DNA sequences capable of mobilization; these sequences are commonly referred to as transposons, transposable elements (TEs), or jumping genes. Although long thought to have no biological significance, advances in DNA sequencing and analytical technologies have enabled precise characterization of TEs and confirmed their ubiquitous presence across all forms of life. These findings have ignited intense debates over their biological significance. The available evidence now supports the notion that TEs exert major influence over many biological aspects of organismal life. Transposable elements contribute significantly to the evolution of the genome by giving rise to genetic variations in both active and passive modes. Due to their intrinsic nature of mobility within the genome, TEs primarily cause gene disruption and large-scale genomic alterations including inversions, deletions, and duplications. Besides genomic instability, growing evidence also points to many physiologically important functions of TEs, such as gene regulation through cis-acting control elements and modulation of the transcriptome through epigenetic control. In this review, we discuss the latest evidence demonstrating the impact of TEs on genome stability and the underling mechanisms, including those developed to mitigate the deleterious impact of TEs on genomic stability and human health. We have also highlighted the potential therapeutic application of TEs.

Detection of structural variations in densely-labelled optical DNA barcodes: A hidden Markov model approach.

Large-scale genomic alterations play an important role in disease, gene expression, and chromosome evolution. Optical DNA mapping (ODM), commonly categorized into sparsely-labelled ODM and densely-labelled ODM, provides sequence-specific continuous intensity profiles (DNA barcodes) along single DNA molecules and is a technique well-suited for detecting such alterations. For sparsely-labelled barcodes, the possibility to detect large genomic alterations has been investigated extensively, while densely-labelled barcodes have not received as much attention. In this work, we introduce HMMSV, a hidden Markov model (HMM) based algorithm for detecting structural variations (SVs) directly in densely-labelled barcodes without access to sequence information. We evaluate our approach using simulated data-sets with 5 different types of SVs, and combinations thereof, and demonstrate that the method reaches a true positive rate greater than 80% for randomly generated barcodes with single variations of size 25 kilobases (kb). Increasing the length of the SV further leads to larger true positive rates. For a real data-set with experimental barcodes on bacterial plasmids, we successfully detect matching barcode pairs and SVs without any particular assumption of the types of SVs present. Instead, our method effectively goes through all possible combinations of SVs. Since ODM works on length scales typically not reachable with other techniques, our methodology is a promising tool for identifying arbitrary combinations of genomic alterations.

Genomic Complexity of Osteosarcoma and Its Implication for Preclinical and Clinical Targeted Therapies.

Osteosarcoma is a genomically complex disease characterized by few recurrent single-nucleotide mutations or in-frame fusions. In contrast, structural alterations, including copy number changes, chromothripsis, kataegis, loss of heterozygosity (LOH), and other large-scale genomic alterations, are frequent and widespread across the osteosarcoma genome. These observed structural alterations lead to activation of oncogenes and loss of tumor suppressors which together contribute to oncogenesis. To date, few targeted therapies for osteosarcoma have been identified. It is likely that effectiveness of targeted therapies will vary greatly in subsets of tumors with distinct key driver events. Model systems which can recapitulate the genetic heterogeneity of this disease are needed to test this hypothesis. One possible approach is to use patient-derived xenograft (PDX) models characterized with regards to their similarity to the human tumor samples from which they were derived. Here we review evidence pointing to the genomic complexity of osteosarcoma and how this is reflected in available model systems. We also review the current state of preclinical testing for targeted therapies using these models.

Robustness encoded across essential and accessory replicons of the ecologically versatile bacterium Sinorhizobium meliloti.

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone.

Rewired RNAi-mediated genome surveillance in house dust mites.

House dust mites are common pests with an unusual evolutionary history, being descendants of a parasitic ancestor. Transition to parasitism is frequently accompanied by genome rearrangements, possibly to accommodate the genetic change needed to access new ecology. Transposable element (TE) activity is a source of genomic instability that can trigger large-scale genomic alterations. Eukaryotes have multiple transposon control mechanisms, one of which is RNA interference (RNAi). Investigation of the dust mite genome failed to identify a major RNAi pathway: the Piwi-associated RNA (piRNA) pathway, which has been replaced by a novel small-interfering RNA (siRNA)-like pathway. Co-opting of piRNA function by dust mite siRNAs is extensive, including establishment of TE control master loci that produce siRNAs. Interestingly, other members of the Acari have piRNAs indicating loss of this mechanism in dust mites is a recent event. Flux of RNAi-mediated control of TEs highlights the unusual arc of dust mite evolution.

Abstract 3603: Megabase-scale determination of complex genetic aberrations of primary cancer genomes at individual DNA molecule resolution

Abstract Cancer is a disease of the genome caused by genetic aberrations such as point mutations and rearrangements. To identify these genetic aberrations, cancer studies rely on DNA sequencing of short reads from highly fragmented DNA molecules that are typically less than 500 bases long. However, genomic structural alterations and context of genetic aberrations are much larger and cannot be effectively analyzed. To characterize such alterations with completeness, the contiguous relationship of variants from a given chromosome homologue must be determined; this analysis process is referred to as genome phasing. In this study, we demonstrate the significant advantages of genome phasing; we utilize a new technology called linked-read sequencing that enables tracking of individual DNA sequence reads to the input high molecular weight DNA at single-molecule resolution through the use of barcoded reads. We then reconstruct at megabase-scale the complex genomic somatic events derived from the original paternal or maternal chromosome homologues and demonstrate new insight into the undiscovered details of cancer genome alterations. We apply this analysis to primary gastrointestinal cancers from surgical resections; our study is a unique achievement given that this type of phased genome sequencing analysis has only been previously conducted on cancer cell lines. For this study, we focus on specific categories of megabase-scale genetic aberrations: (1) copy number variants (CNV) derived from segmental duplications, (2) structural rearrangements, and (3) aneuploidy. As an example from gastric cancer metastases, we identified a set of unique and site-specific FGFR2 amplification events corresponding to segmental duplications. The structural details of this amplification were only evident using barcoded sequence reads and phased analysis. Secondly, in a primary colorectal adenocarcinoma we resolved a complex recombination event that covered the majority of the chromosome 5 q-arm. It contained a set of complex rearrangements that are difficult to determine by short read sequencing but can be identified using linked reads. This rearrangement event arose from a combination of a 80Mb homologous recombination that contained a small interval ∼2.6Mb deletion. The allelic loss of two candidate cancer drivers, SMAD5 and TGFB, occurred in the affected interval. Finally, to assess aneuploidy we relied on the imbalanced ratios between paternal and maternal chromosomal homologues to generate contiguous blocks of variants on the order of tens and up to hundreds of megabases long. We performed this type of analysis on a number of cancer genomes derived from primary colorectal adenocarcinomas, and were able to robustly determine the phased single nucleotide variant content and large-scale genomic alterations involving entire chromosome arms. Citation Format: Billy Lau, John Bell, Stephanie Greer, Grace Zheng, Christina Wood, Hanlee Ji. Megabase-scale determination of complex genetic aberrations of primary cancer genomes at individual DNA molecule resolution. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3603.

X chromosome aneuploidy in the Alzheimer’s disease brain

BackgroundAlthough the link between brain aging and Alzheimer’s disease (AD) is a matter of debate, processes hallmarking cellular and tissue senescence have been repeatedly associated with its pathogenesis. Here, we have studied X chromosome aneuploidy (a recognized feature of aged cell populations) in the AD brain.ResultsExtended molecular neurocytogenetic analyses of X chromosome aneuploidy in 10 female AD as well as 10 age and sex matched female control postmortem brain samples was performed by multiprobe/quantitative FISH. Additionally, aneuploidy rate in the brain samples of 5 AD and as 5 age and sex matched control subjects were analyzed by interphase chromosome-specific multicolor banding (ICS-MCB). Totally, 182,500 cells in the AD brain and 182,500 cells in the unaffected brain were analyzed. The mean rate of X chromosome aneuploidy in AD samples was approximately two times higher than in control (control: mean - 1.32%, 95% CI 0.92- 1.71%; AD: mean - 2.79%, 95% CI 1.88-3.69; P = 0.013). One AD sample demonstrated mosaic aneuploidy of chromosome X confined to the hippocampus affecting about 10% of cells. ICS-MCB confirmed the presence of X chromosome aneuploidy in the hippocampal tissues of AD brain (control: mean - 1.74%, 95% CI 1.38- 2.10%; AD: mean - 4.92%, 95% CI 1.14-8.71; P < 0.001).ConclusionsAddressing X chromosome number variation in the brain, we observed that somatically acquired (post-zygotic) aneuploidy causes large-scale genomic alterations in neural cells of AD patients and, therefore, can be involved in pathogenesis of this common neurodegenerative disorder. In the context of debates about possible interplay between brain aging and AD neurodegeneration, our findings suggest that X chromosome aneuploidy can contribute to both processes. To this end we conclude that mosaic aneuploidy in the brain is a new non-heritable genetic factor predisposing to AD.

Next Generation Sequencing-Based Molecular Dissection Of Lineage-Specific Mutational Hierarchies In Oligoclonal Primary and Xenografted Myelodysplasia

BackgroundThe underlying molecular defects in myelodysplastic syndromes (MDS), which are a heterogeneous group of malignant clonal hematologic disorders, are not well understood. Recently, next generation sequencing (NGS) based whole genome and exome sequencing highlighted the oligoclonal nature of persistent MDS clones that are present already at early disease stages. The reconstruction of mutational hierarchies in MDS clones and distinction of primary founder from subsequently acquired lesions has yet to be thoroughly interrogated and is likely to aid dissecting the molecular pathogenesis of MDS. MethodsAn amplicon-based NGS assay using the Roche 454 GS Junior system was established within the IRON-II framework study in order to screen for 17 commonly mutated genes in MDS. Genomic DNA from purified mononuclear bone marrow (BM) cells of 23 MDS IPSS low/int1 risk subjects was screened for somatic mutations. Called variants were compared to dbSNP and COSMIC database entries to rule out germline polymorphisms. In addition, copy number variation analysis was performed by Affymetrix SNP 6.0 array profiling. Custom pyrosequencing assays and interphase-FISH were applied for sensitive quantification of lesion burdens in FACS-sorted myeloid, erythroid, lymphoid and stem/progenitor cells. These were isolated from patients' primary BM as well as their long-term engrafted human xenotransplants using our recently established MDS xenograft model. ResultsIn this work, we identified 12 oligoclonal BM samples with ≥2 molecular lesions. Of note, varying frequencies of individual mutations between different sorted cell subsets from primary or human xenografted BM support the notion that distinct MDS (sub-)clones from these subjects contributed to hematopoiesis simultaneously and lead to differential engraftment between xenografts. Comparison of variable subset-specific mutation burdens allowed deciphering the individual hierarchical architecture of the mutational landscape from 9 individuals. ASXL1, SF3B1 and SRSF2 were detected as a primary lesion for 2 patients each. In contrast, large-scale genomic alterations such as del(5q), del(RUNX1) or trisomy 8 occurred as late-end lesion or even defined distinct clones which coexist with others harboring different mutations as detected for 2 subjects. Surprisingly, CD19+ and CD3+ lymphocytes from primary and/or xenografted BM displayed significant mutational burden of at least 1 mutation in 50% of the MDS cohort (5/10). Moreover, mutations were detected simultaneously in lymphocytes (hCD19+) as well as myeloid (hCD33+) and erythroid (hCD235a+) cells from three xenografted samples indicating a potent multilineage engraftment capability of MDS hematopoietic stem cells.Interestingly, one individual presented with high RUNX1 mutational frequency in the primary early progenitor fraction (CD34+CD38+), which was absent in the stem-cell enriched fraction (CD34+CD38-), whereas TET2, ZRSR2 and ASXL1 mutations were detected in both fractions and their xenografts. Intriguingly, only xenotransplantation of primary CD34+38- BM cells lead to long-term engraftment of RUNX1 wild type human BM cells in mice, while CD34+CD38+ BM cells gave rise to short term engraftment of RUNX1 mutated human BM cells indicating that mutated RUNX1might originate in a more committed progenitor fraction with limited self-renewal potential. ConclusionMolecular characterization of oligoclonal mutation patterns in primary and xenograft BM allowed the establishment of individual mutational hierarchies and indicates a relatively random order in the mutational evolution of MDS clones, although spliceosome mutations appear as rather early events. Furthermore, our analysis revealed engraftment of independent MDS clones in different mice xenografted with the same subject material, which opens the door to the in vivo study of isolated clones with respect to their pathomechanisms and response to treatment. Our data also suggests that the occurrence of large-scale genomic aberrations is frequently preceded by small-scale gene mutations, emphasizing their potential role in disease diagnosis and risk stratification. Finally, detection of MDS specific mutations in the lymphocytic compartment might be involved in facilitating impaired immune functionality and needs to be investigated prospectively. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Staller:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria.

Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria.

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins form adaptive immune systems in bacteria to combat phage and other foreign genetic elements. Typically, short spacer sequences are acquired from the invader DNA and incorporated into CRISPR arrays in the bacterial genome. Small RNAs are generated that contain these spacer sequences and enable sequence-specific destruction of the foreign nucleic acids. Occasionally, spacers are acquired from the chromosome, which instead leads to targeting of the host genome. Chromosomal targeting is highly toxic to the bacterium, providing a strong selective pressure for a variety of evolutionary routes that enable host cell survival. Mutations that inactivate the CRISPR-Cas functionality, such as within the cas genes, CRISPR repeat, protospacer adjacent motifs (PAM), and target sequence, mediate escape from toxicity. This self-targeting might provide some explanation for the incomplete distribution of CRISPR-Cas systems in less than half of sequenced bacterial genomes. More importantly, self-genome targeting can cause large-scale genomic alterations, including remodeling or deletion of pathogenicity islands and other non-mobile chromosomal regions. While control of horizontal gene transfer is perceived as their main function, our recent work illuminates an alternative role of CRISPR-Cas systems in causing host genomic changes and influencing bacterial evolution.

Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

Abstract 826: SCL8A1 negatively regulated by miR-223 is one candidate for sodium/calcium transportation and low level of apoptosis in penile carcinomas.

Abstract Background: Penile cancer (PeCa) is a rare disease in developed countries, but with high incidence in South America and Africa. Surgery is frequently used for local control of the disease and can lead to partial or complete amputation of the organ. Integrative analysis of large-scale gene expression and genomic alterations in PeCa showed down-expression of the solute carrier family 8 (SLC8A1, sodium/calcium exchanger) with no genomic changes suggesting that other mechanisms, such as micro-RNAs, could be involved in gene regulation. Intracellular Ca2+ can contribute to cancer cell proliferation and tumor progression, but can also induce tumor apoptosis by calcium-regulated cell death pathways. Therefore, we speculated if the down-expression of SLC8A1 mediated, at least in part, by miR-223 in penile carcinomas could lead to a reduction of cellular calcium levels and apoptosis suppression and to contribute for increased cell proliferation. Aim: This study aimed to confirm the gene expression patterns of SLC8A1 and miR-223 in PeCa and normal tissues and to evaluate calcium levels and apoptosis in in a subset of the samples showing these alterations. Methods: RNA was obtained from 39 fresh PeCa and 4 normal penile tissues. SLC8A1 gene and miRNA-223 transcript levels were evaluated by RT-qPCR. Fresh PeCa (n=2) matched with adjacent (n=1) and normal tissue from autopsy (n=1) showing alterations of SLC8A1 and miRNA-233 were selected and evaluated for calcium distribution by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). Caspase-3 and Ki-67 were evaluated by immunohistochemistry in 9 selected PeCa. Results: Significant down-expression of SLC8A1 (P=0.010) and overexpression of miR-223 (P=0.0072) was detected in tumors in the comparison with normal controls. A significant negative correlation was detected between SLC8A1 transcript levels and miR-223 (P=0.015, r = -0.482), suggesting that this gene is regulated by this miRNA. LA-ICP-MS mapping revealed decreased calcium levels in tumor cells compared with adjacent normal cells and also compared with a normal tissue. Penile carcinoma tissue showed lower calcium counts (Ca44/C13 ratio=5,14 +/- 0,81) compared with normal tissues (Ca44/C13 ratio=6,34 +/- 4,63). In addition, PeCa presented considerably higher sodium counts (counts per second = 6.5 x 10ˆ5) compared with normal tissues (counts per second = 4 x 10ˆ5). Decreased caspase-3 and increased Ki-67 levels were detected in tumor tissues, indicating that low-level apoptosis and high cell proliferation index occurred in these samples. Conclusions: The results suggested that down-regulation of SLC8A1 gene probably mediated by miR-223 can contribute to calcium levels reduction in PeCa and consequently in apoptosis suppression of these cells. Further functional studies are needed to prove this hypothesis. Citation Format: Juan J. Muñoz, Sandra A. Drigo, Mateus C. Barros, Fábio Marchi, Fernando A. Soares, Gustavo Pessoa, Gustavo Pessoa, José Vassallo, Marco A. Arruda, Silvia R. Rogatto. SCL8A1 negatively regulated by miR-223 is one candidate for sodium/calcium transportation and low level of apoptosis in penile carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2013-826

Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer.

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

Infantile Convulsions with Paroxysmal Dyskinesia (ICCA Syndrome) and Copy Number Variation at Human Chromosome 16p11

BackgroundBenign infantile convulsions and paroxysmal dyskinesia are episodic cerebral disorders that can share common genetic bases. They can be co-inherited as one single autosomal dominant trait (ICCA syndrome); the disease ICCA gene maps at chromosome 16p12-q12. Despite intensive and conventional mutation screening, the ICCA gene remains unknown to date. The critical area displays highly complicated genomic architecture and is the site of deletions and duplications associated with various diseases. The possibility that the ICCA syndrome is related to the existence of large-scale genomic alterations was addressed in the present study.Methodology/Principal FindingsA combination of whole genome and dedicated oligonucleotide array comparative genomic hybridization coupled with quantitative polymerase chain reaction was used. Low copy number of a region corresponding to a genomic variant (Variation_7105) located at 16p11 nearby the centromere was detected with statistical significance at much higher frequency in patients from ICCA families than in ethnically matched controls. The genomic variant showed no apparent difference in size and copy number between patients and controls, making it very unlikely that the genomic alteration detected here is ICCA-specific. Furthermore, no other genomic alteration that would directly cause the ICCA syndrome in those nine families was detected in the ICCA critical area.Conclusions/SignificanceOur data excluded that inherited genomic deletion or duplication events directly cause the ICCA syndrome; rather, they help narrowing down the critical ICCA region dramatically and indicate that the disease ICCA genetic defect lies very close to or within Variation_7105 and hence should now be searched in the corresponding genomic area and its surrounding regions.

Somatic Genome Variations in Health and Disease

It is hard to imagine that all the cells of the human organism (about 1014) share identical genome. Moreover, the number of mitoses (about 1016) required for the organism’s development and maturation during ontogeny suggests that at least a proportion of them could be abnormal leading, thereby, to large-scale genomic alterations in somatic cells. Experimental data do demonstrate such genomic variations to exist and to be involved in human development and interindividual genetic variability in health and disease. However, since current genomic technologies are mainly based on methods, which analyze genomes from a large pool of cells, intercellular or somatic genome variations are significantly less appreciated in modern bioscience. Here, a review of somatic genome variations occurring at all levels of genome organization (i.e. DNA sequence, subchromosomal and chromosomal) in health and disease is presented. Looking through the available literature, it was possible to show that the somatic cell genome is extremely variable. Additionally, being mainly associated with chromosome or genome instability (most commonly manifesting as aneuploidy), somatic genome variations are involved in pathogenesis of numerous human diseases. The latter mainly concerns diseases of the brain (i.e. autism, schizophrenia, Alzheimer’s disease) and immune system (autoimmune diseases), chromosomal and some monogenic syndromes, cancers, infertility and prenatal mortality. Taking into account data on somatic genome variations and chromosome instability, it becomes possible to show that related processes can underlie non-malignant pathology such as (neuro)degeneration or other local tissue dysfunctions. Together, we suggest that detection and characterization of somatic genome behavior and variations can provide new opportunities for human genome research and genetics.