Clinical Genomic Analysis Workshop 2013

Abstract: Chromosomal instability is a hall mark of cancer. In early stages of cancer development the instability is caused by stress on the DNA replication. However, the molecular basis for this replication perturbation remained unknown. We have studied the replication dynamics in cells enforced to proliferate by aberrant activation of the Rb-E2F pathway, due to over-expression of the viral (HPV-16 E6/E7) or cellular (cyclin E) oncogenes. This enforced cell proliferation with an insufficient pool of nucleotides to support normal DNA replication resulting in replication perturbation, DNA damage and genome instability. Importantly, an exogenous supply of nucleosides rescued the replication stress, decreased the replication-induced DNA damage and reduced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-Myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Hence, the low-nucleotide pool is a result of unbalanced activation of nucleotide biosynthesis genes.

Tumorigenicity is driven by alterations in cellular and environmental factors. We further analyzed the effect of folate, an environmental factor essential for nucleotide biosynthesis, on the early stages of cancer. We show that suboptimal levels of folate, which are associated with increased risk of cancer development, lead to concentration-dependent replication-induced DNA damage. Importantly, folate deficiency significantly enhances the replication stress caused by aberrant oncogene expression, leading to significantly increased DNA damage and tumorigenic potential. These findings shed new light on the combined effect of cellular and environmental factors on cancer and indicate that the extent of nucleotide-driven replication stress is a key regulator of tumorigenicity.

Bio: TBD

Abstract: Cancer is, at the cellular level, an evolutionary, adaptive process in which cells acquire new proliferative and invasive capabilities. The cancer evolutionary process depends on genetic variation that is generated by somatic mutation. While, much focus has been given to identifying specific positively selected cancer 'driver' mutations, not much is currently understood about the more general dynamics of how natural selection affects cancer somatic mutations. We have been using extensive data of somatic substitutions from a large number of breast tumors, gathered by the Cancer Genome Atlas project, to characterize the intensity with which purifying and positive selection affect somatic mutations during the evolution of these tumors. We demonstrate that breast tumor somatic mutations are subject to extremely relaxed purifying selection. At the same time, BRCA somatic mutations are also strongly influenced by positive selection, which affects most strongly genes that are globally expressed across tissues.

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Abstract: In the past two decades chromosomal micro arrays (CMA) have emerged as a powerful tool for identifying novel genomic syndromes. This technology (using different platforms) allows the identification of sub-microscopic chromosomal deletions and duplications that are 100 times smaller than those detected by the traditional karyotype. Our one decade experience utilizing this technology both in the prenatal and postnatal settings, clearly suggests that changes in the dosage of the DNA material makes up part of the normal variation in the general healthy population, as well as being a common cause for numerous syndromes.

The challenges related to the "learning curve" primarily regards our need to differ pathogenic vs non-pathogenic copy number variants (CNVs) identified by CMA. The relationship of genotype to phenotype for CNVs is rarely as simple as the dominant and recessive patterns described by Mendel, with some known and others ever defined. The puzzle is just beginning to reveal its full magnitude. The clinical usage of CMA technology as a routine tool for the work-up of patients, families and during pregnancies is essential both for researchers and clinicians attempting to interpret the potential pathogenicity of each identified CNV. In the genomic era, when new technologies such as massive parallel sequencing emerge, insights from CMA is becoming a powerful diagnostic tool aiming to affect treatment modalities of human diseases.

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Abstract: In this talk I will review various possible designs for clinical trials involving genetic markers, and address their advantages and disadvantages. I will also discuss the possibility of combining a PGx trial within a "regular" clinical trial, and the additional considerations needed for such a trial.

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Abstract: Understanding viral infections in clinical samples is critical for effective patient health care. The study of a transcriptome is a powerful tool for perfecting gene expression and RNA-based regulation in any organism. Our laboratory uses massive parallel sequencing technologies (Deep sequencing or Next Generation Sequencing) that generate billions of reads per experiment, to study the RNA-mediated regulatory mechanisms during cellular processes. We are currently focusing on emerging principles controlling non-coding RNA-mediated regulatory mechanisms during pathogenesis caused by viral infection. Understanding the impact of this regulation on viral life cycles together with host interactions, might provide insight into future novel targets for intervention. Our genome-wide analyses will bring us closer towards having a full non-coding RNA 'signature map' during infection from both the viral and host perspectives. These findings will make significant contribution in the field of systems pathogenicity.

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Abstract: The ubiquitin and ubiquitin-like (Ubl) modification pathways regulate various aspects of cellular and physiological processes in health and disease. However, studies mapping different targets of Ubls on a genome-wide scale are still lacking. We developed a novel high-throughput method for detection of post-translational modifications using an activity-based assay. We employed the PTM profiling approach to identify the substrates of ubiquitin and six additional Ubl modifiers (e.g. Ubiquitin, SUMO, NEDD8, FAT10, UFM1 and ISG15) in mitosis. We identified over 1500 potential targets (known as well as novel ones) of ubiquitin and Ubl modifications. Our analysis revealed highly independent target specificities for the different Ubl modifiers where between 100-200 specific proteins distinctly classify each of these pathways (i.e. were reactive exclusively toward one Ubl). We mapped different molecular functions and biological processes to each Ubl pathway and revealed novel specialized roles in various biological processes.

Interestingly, FAT10 exhibited the most significant effect in target modification upon release from mitotic arrest. Indeed, we found that inhibiting the FAT10 pathway resulted in a mitotic arrest that ultimately lead to cell death. This data is supported by upregulation of FAT10 in different types of cancer, suggesting an underappreciated and important role in mitotic regulation and malignancy.

Finally, PTM profiling establishes a broadly-applicable and systematic approach to study regulation of PTMs in the context of human diseases and offers a new molecular dimension for clinical diagnostics.

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Abstract: Since last two decades plethora of the data is generated on gene polymorphism and point mutations. Special attention has been given to search any linkage between susceptibility to infectious diseases and single nucleotide polymorphisms (SNPs) in genes of immune system like Toll like receptors (TLRs), MHC genes, defensins, complement system etc. The first part of the presentation will be dedicated to a review of known SNPs in various genes of immune system and their linkage to the susceptibility to infectious diseases. The second part will be focused on novel mutations in the Toll like receptor -1 (TLR1) and TLR2 of human and animal, responsible for increase in the susceptibility to mycobacterial infection. Human and animal TLR1 consist of 20 Lucine rich repeats (LRRs) that take part in the mycobacterial antigen recognition. The central region of the extracellular domain of human TLR1 (LRR9 to LRR12) is necessary for the sensing of bacterial lipopeptides. We observed that the central part of TLR1 ectodomain (LRR9 to LRR11) is more irregular and prone to missense mutations. Further we showed that novel mutation Val220Met in LRR10 motif at the 9th amino acid position disrupts hydrogen bonds in the LRR loop structure that cause reduced recognition of mycobacteria. In the case of TLR2, the TIR domain is crucial as it forms a TIR-TIR dimerized platform (TLR1-TLR2 and TLR2-TLR6), which promotes homotypic protein-protein interactions and further downstream signaling necessary to fight against mycobacteria. We found that homozygous 670Leu mutation causes impaired dimerization of TLR2-TIR domain with its counterparts and thus cause abrupt downstream signaling. Mapping of such mutations in the population, finding their linkage with various infectious diseases and summarizing plethora of the generated data from such experiments in a systematic way is challenging but make a worthwhile contribution to clinical immonogenetics.

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Abstract: During brief epileptic bursts, principal neurons fire together for tens to hundreds of milliseconds, producing a large extracellular potential ("field"). Superimposed on this large field are high-frequency oscillations, from ~100 to several hundred Hz. Two distinctive means of coupling between neurons cooperate to generate the event. Recurrent excitatory synaptic connections shape the overall event, but gap junction coupling (between pyramidal cells) produces the fast oscillations. I will describe the dissection of the cellular mechanisms via in vitro experiments (on rodent and human tissue) and via computer modeling and network theory. Experimentally, the fast oscillations can be evoked alone, during blockade of chemical synapses; but blockade of gap junctions abolishes BOTH the fast oscillations and the larger burst. Other lines of evidence pointing toward a critical role for gap junctions in epilepsy-related very fast oscillations are these: a) large-scale spatial patterns of cortical fast oscillations, resembling an excitable medium; b) the existence of "glissandi" (~30 to >150 Hz) oscillations in epileptic tissue, with chemical synapses blocked; c) recent data showing that fast ripples (>250 Hz) in resected human tissue persist without chemical synapses. These data suggest that a targeted manipulation of selected gap junctions might prevent certain seizure events.

Bio: TBD